The  Organic  Matter  of  the  Soil:    A  Study  of 

the  Nitrogen  Distribution  in  Different 

Soil  Types 


A  THESIS  SUBMITTED   TO  THE   FACULTY  OF  THE  GRADUATE 
SCHOOL  OF  THE  UNIVERSITY  OF,  MINNESOTA 


BY 


CLARENCE  AUSTIN ,  MORROW,  B.S.,M.A. 

IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF  DOCTOR  OF  PHILOSOPHY 


JUNE,  1918 


MINNEAPOLIS 
UNIVERSITY  PRINTING  CO. 


EXCHANGE 


The  Organic  Matter  of  the  Soil:    A  Study  of 

the  Nitrogen  Distribution  in  Different 

Soil   Types 


A  THESIS   SUBMITTED   TO   THE   FACULTY   OF   THE   GRADUATE 
SCHOOL  OF  THE  UNIVERSITY  OF  MINNESOTA 

BY 

CLARENCE  AUSTIN  MORROW,  B.S.,  M.A. 

<  i 

IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF  DOCTOR  OF  PHILOSOPHY 

JUNE,  1918 


MINNEAPOLIS 
UNIVERSITY  PRINTING  CO. 


TO  MY  FATHER  AND  MOTHER 


428979 


ACKNOWLEDGMENT. 

This  investigation  was  carried  out  under  the  direction  of  Doc- 
tor Ross  Aiken  Gortner.  The  author  takes  this  opportunity  to 
express  his  appreciation  and  gratitude  for  the  help  extended  during 
the  prosecution  of  this  work,  and  for  the  unfailing  kindness, 
thoughtful  advice  and  encouragement  which  was  given. 

C.  A.  M. 

University    of   Minnesota. 
Division  of  Soils. 
January  1917. 


TABLE   OF  CONTENTS. 

Page 
I.     Introduction:     Our  present  knowledge  of  the  organic   matter  of 

the  soil 7 

A.  Early  investigations ^ 7 

B.  The  humus  theory  of  Grandeau 12 

C.  The  complexity  of  the  ammonia  soluble   material 14 

D.  The  presence  of  definite  organic  compounds  in  the  soil..  15 

E.  The  origin  of  organic  compounds  in  the  soil 16 

1.  Nucleoprotein    decomposition 17 

2.  Nucleic    acid    decomposition 17 

3.  Lecithin  decomposition 18 

F.  Bacterial    processes    which    influence    the    form    of    soil 

nitrogen  19 

1.  Deamination   or   reduction , 20 

2.  Decarboxylation   or  amine   formation 20 

3.  Hydrolysis 20 

4.  Oxidation 21 

G.  'Nitrogen  distribution  in  the  soil . .  . . 21 

H.     A   summary  of  the  nature   of  the  organic  matter   of  the 

soil  in  the  light  of  our  present  knowledge 29 

II.     Experimental:     A  study  of  the  nitrogen  distribution  in  different 

soil   types 31  - 

A.  The  problem 31 

B.  The  material : 31 

1.  Calcareous   black   grass-peat 31 

2.  Sphagnum-covered  peat 31 

3.  Acid  "muck"  soil 31 

4.  Fargo   clay   loam 32 

5.  Fargo  silt  loam 32 

6.  Carrington   silt   loam 32 

7.  Hempstead  silt  loam 32 

8.  Prairie-covered   loess 32 

9.  Forest-covered  loess 32 

10.     Hempstead  silt  loam   subsoil 32 

C.  The  method 33 

1.  The  method  in  detail  for  a  peat  soil 34 

2.  The  method  in  detail  for  a  mineral  soil 36 

3.  The  method    for    determination    of   "Jodidi    num- 

bers"   ; 37 

4.  The  determination   of   nitrogen 38 

4.     The   analytical   data 38 

1.  The  analysis  of  "fibrin  from  blood"  hydrolyzed  in 

the  presence  of  100  grams  of  ignited  subsoil  38 

2.  Calcareous   black  grass-peat 41 

3.  Sphagnum-covered  peat 42 

4.  Acid    "muck"    soil..  42 


5.  Fargo  clay  loam 43 

6.  Fargo  silt  loam 44 

7.  Carrington  silt  loam 45 

8.  Hempstead  silt  loam 45 

9.  Prairie-covered   loess 46 

10.  Forest-covered  loess 47 

11.  Sphagnum-covered   peat   hydrolyzed   in    the   pres- 

ence of  nine  times  its  weight  of  a  mineral 
subsoil  47 

12.  Sphagnum-covered   peat   hydrolyzed   in   the   pres- 

ence of  metallic  tin 49 

13.  Analysis  of  a  1  per  cent,  hydrochloric  acid  extract 

of  sphagnum-covered  peat  and  (in  part)  of 
calcareous  black  grass-peat 50 

14.  Analysis  of  a  portion  of  sphagnum-covered  peat 

soluble  in  4  per  cent,  sodium  hydroxide  and 
precipitated  by  hydrochloric  acid  and  (in 
part)  of  a  similar  solution  from  a  calcareous 
black  grass-peat 52 

15.  Analysis  of  a  portion  of  sphagnum-covered  peat 

soluble  in  4  per  cent  sodium  hydroxide  and 
not  precipitated  by  hydrochloric  acid  and  (in 
part)  of  a  similar  solution  from  a  calcareous 
black  grass-peat 54 

16.  "Jodidi   numbers"    ! 55 

17.  Summary  tables 57 

18.  An  attempt  to  isolate  pure  proteins  from  a  soil     59 

a.  Extraction  with  70  per  cent  ethyl  alcohol     59 

b.  Extraction  with  absolute  alcohol 6U 

c.  Extraction  with  10  per  cent  sodium  chlor- 

ide       61 

III.     Discussion   62 

A.  Changes  in  nitrogen  distribution  in   a   protein  when   hy- 
drolyzed in  the  presence  of  a  mineral  soil 62 

B.  The  human  nitrogen,  its  origin,  and  significance 63 

C.  The  effect  of  the  quantity  of  acid  used  for  the  hydrolysis 
on    the   amount   of   nitrogen    dissolved    and   the    nitrogen 
distribution  in  soils    66 

D.  The  percentage   of  soil   nitrogen   extracted   by   acid   hy- 
drolysis          66 

E.  "Jodidi    numbers" 67 

F.  Attempts   to   extract    proteins   from   the    soil 67 

G.  A  consideration  of  the  nitrogen  distribution  in   different 
extracts    from    the    sphagnum-covered    peat 67 

H.     General  conclusions  in  regard  to  the  distribution  of  soil 

nitrogen  in  different  soil  types 68 

IV.     Summary     70 

V.     Literature  cited 72 

Biographical 80 


I.     INTRODUCTION:     OUR  PRESENT   KNOWLEDGE   OF 
THE  ORGANIC  MATTER  OF  THE  SOIL. 

A.     Early  Investigations. 

It  has  been  recognized  from  the  time  of  the  alchemists  that 
manures  are  of  fundamental  importance  for  the  growth  of  plants. 
The  alchemists  believed  that  by  a  process  of  transmutation  water 
was  converted  into  plant  tissue.  In  an  attempt  to  prove  this  an 
interesting  experiment  was  conducted  by  van  Helmont  (1648). 
In  a  large  earthen  vessel  he  placed  200  pounds  of  dry  earth,  and 
planted  in  it  a  small  willow  tree  weighing  five  pounds,  and  for  five 
years  he  watered  the  plant  either  with  rain  or  distilled  water.  .At 
the  end  of  that  time  he  pulled  up  the  willow  and  found  that  it 
weighed  169  pounds  and  three  ounces.  The  dry  soil  remaining  after 
the  experiment  was  found  to  have  lost  only  two  ounces.  He  drew 
the  apparently  justifiable  conclusion  that  164  pounds  of  roots,  bark, 
leaves,  and  branches  had  been  produced  by  direct  transmutation 
of  water. 

It  is  evident  that  it  was  essential  to  establish  the  composition 
of  water  and  some  of  the  components  of  the  air  before  further 
work  could  have  real  value. 

Not  until  the  discovery  of  oxygen  by  Scheele  (1777)  and 
the  proof  of  the  composition  of  water  by  Cavendish  (1784),  as  well 
as  the  work  of  de  Saussure  (1804)  regarding  the  role  played  by 
carbon  dioxide  in  plant  and  animal  life,  did  we  have  any  real 
knowledge  concerning  the  sources  of  matter  stored  up  in  plants. 

During  the  first  quarter  of  the  nineteenth  century  organic  com- 
pounds were  regarded  as  capable  of  being  synthesized  only  in  the 
living  cells  of  plants  or  animals.  This  idea  that  organic  compounds 
could  be  formed  only  through  a  special  vital  force  was  overthrown 
by  the  classic  work  of  Wohler  (1828)  when  he  prepared  urea,  a 
purely  animal  product*,  by  evaporating  ammonium  cyanate  to 
dryness.  This  fact  attracted  the  attention  of  chemists,  and  prac- 
tically all  work  done  from  that  time  until  1840  was  on  some  phase 
of  organic  chemistry.  This  overthrow  of  the  belief  in  a  vital  force 
and  the  improved  method  of  organic  analysis  by  Berzelius  (1808- 
18)  paved  the  way  for  a  more  thorough  understanding  of  the  part 
taken  by  the  organic  matter  in  the  soil,  and  consequently  created 
a  renewed  interest  in  scientific  investigations  relating  to  agricul- 
ture. 

Some  important  work  had  been  accomplished  previous  to  this 
time.  The  most  valuable  was  that  of  de  Saussure's  (1804)  "Re- 
cherches  chimiques  sur  la  vegetation"  which  was  the  first  syste- 
matic work  showing  the  source  of  compounds  stored  up  in  the 
plant.  He  pointed  out  that  the  quantitative  increase  in  the  carbon, 
hydrogen,  and  oxygen,  when  plants  were  exposed  to  sunlight,  was 

*Fosse    (1912)    notes  the   presence  of  urea  in   certain   plants. 


8 

due  to  the  carbon  dioxide  of  the  air  and  water  of  the  soil.  He  be- 
lieved that  the  nitrogen  of  the  soil  was  the  chief  source  of  the 
nitro-gen  found  in  plants.  Unfortunately  his  conclusions  were 
not  accepted  at  that  time,  and  it  was  not  until  about  fifty  years 
later,  when  other  investigators  had  repeated  his  experiments,  that 
his  results  were  finally  accepted  by  botanists  and  chemists. 

One  of  the  first  investigators  to  see  the  relation  between  chem- 
istry and  agriculture  was  that  of  Sir  Humphry  Davy  (1813),  who 
published  a  book  entitled,  "Elements  of  Agricultural  Chemistry." 
This  treated  of  the  composition  of  air,  soil,  manures,  plants,  and 
of  the  influence  of  heat  and  light  upon  the  growth  of  plants. 

Thaer  (1809-10)  contended  that  humus  determined  the  fertility 
of  the  soil,  that  plants  obtained  their  food  mainly  from  humus, 
and  that  the  carbon  compounds  of  plants  were  produced  from  the 
organic  carbon  compounds  of  the  soil.  These  ideas  gave  rise  to  his 
so-called  humus  theory,  which  was  later  shown  to  be  inadequate. 
His  writings,  however,  did  much  to  stimulate  later  investigation. 

The  French  investigator  Boussingault  verified  much  of  the 
earlier  work  of  de  Saussure  and  secured  many  additional  facts 
concerning  the  chemistry  of  growth.  His  predecessors  had  sought 
to  solve  the  question  as  to  whether  plants  assimilate  the  free  or 
uncombined  nitrogen  of  the  atmosphere.  Boussingault  (1838, 
1838a)  improved  the  methods  for  the  determination  of  the  point  in 
question,  and  showed  that  peas  and  clover  could  get  their  nitrogen 
from  the  air  while  wheat  could  not.  Unfortunately,  he  did  not 
make  as  much  of  this  discovery  as  he  might  have  done. 

Boussingault  was  the  first  to  have  a  chemical  laboratory  lo- 
cated on  a  farm  and  to  make  investigations  along  a  practical  line 
in  connection  with  agriculture.  His  was  the  first  agricultural  ex- 
periment station. 

The  investigations  of  de  Saussure,  Boussingault,  Davy,  Thaer, 
and  others  paved  the  way  for  the  work  and  writings  of  Liebig.  He 
published  (1840)  "Organic  Chemistry  in  its  Application  to  Agri- 
culture and  Physiology,"  which  was  an  important  factor  in  at- 
tracting the  attention  of  the  public  to>  agricultural  problems.  Many 
of  his  investigations  and  discoveries  in  the  field  of  organic  chem- 
istry were  applied  directly  to  his  interpretation  of  these  problems. 

He  assailed  the  humus  theory  of  Thaer  and  showed  that  humus 
could  not  be  an  adequate  source  of  the  plant's  carbon.  By  applying 
the  exact  methods  of  chemistry  to  agriculture  Liebig  succeeded  in 
establishing  that  plants  derive  the  carbon  of  their  tissues  from 
the  carbon  dioxide  of  the  air,  and  not  from  the  carbon  compounds 
that  may  be  present  in  the  soil.  He  came  to  regard  the  ammonia 
of  the  air  as  analogous  with  the  carbon  dioxide  of  the  air,  and 
preached  the  doctrine  that  plants  were  able  to  derive  their  nitrogen- 
ous food  from  the  atmosphere.  In  the  Farmer's  Magazine,  for  in- 
stance, he  writes : 

If  the  soil  be  suitable,  if  it  contains  a  sufficient  quantity  of  alkalis,  phos- 
phates, and  sulphates,  nothing  will  be  wanting.  The  plants  will  derive  their 
ammonia  from  the  atmosphere  as  they  do  carbonic  acid.  (Cited  by  Russell, 
1912.) 

Although  the  work  of  Liebig  was  not  conducted  in  connection 
with  field  experiments,  it  had  a  stimulating  effect  upon  agricultural 


investigation,  and  we  are  greatly  indebted  to  him  for  summarizing 
previous  work  and  pointing  out  valuable  lines  of  future  research. 
In  his  book  (1840)  he  states,  that— 

a  rational  system  of  agriculture  cannot  be  formed  without  the  application 
of  scientific  principles,  for  such  a  system  must  be  based  on  an  exact  ac- 
quaintance with  the  means  of  nutrition  of  vegetables,  and  with  the  influence 
of  soils,  and  actions  of  manures  upon  them.  This  knowledge  we  must  seek 
from  chemistry,  which  teaches  the  mode  of  investigating-  the  composition  and 
study  of  the  character  of  the  different  substances  from  which  plants  derive 
their  nourishment. 

In  one  essential  point,  however,  he  fell  into  error.  Lawes,  the 
pioneer  experimenter  on  agriculture  in  England,  flatly  denied  the 
accuracy  of  Liebig's  conclusions  as  regards  nitrogen  assimilation. 
The  results  of  the  investigations  at  Rothamsted  as  conducted  by 
Lawes  and  Gilbert  (1851)  on  the  non-assimilation  of  atmospheric 
nitrogen  by  crops,  were  accepted  as  conclusive  evidence  upon  this 
much  discussed  question. 

The  alkali  soluble  portion  of  the  organic  matter  of  the  soil 
has  formed  for  many  years  the  subject  of  keen  interest  and  discus- 
sion. This  portion  of  the  soil  organic  matter  was  called  "humus" 
by  the  earlier  writers,  but  this  name  has  in  more  recent  times  been 
used  by  some  American  and  most  European  investigators  to  desig- 
nate the  total  organic  matter  of  the  soil.  I  have  used  the  term 
throughout  this  paper  in  its  original  meaning.  In  early  days  the 
"humus"  was  regarded  as  being  of  very  simple  composition.  De 
Saussure  (1804)  for  instance,  described  it  as  a  "brown  combustible 
powder  soluble  in  alkalies  and  ammonia  compounds." 

Klaproth*  applied  the  name  "ulmin"  to  dark  colored  amorphous 
bodies  such  as  those  obtained  by  Vauquelin  (1797)  from  the  bark 
of  diseased  elm  trees.  Sprengel  (De  Candolle**  1833,  p.  280),  who 
obtained  similar  bodies  from  soils  applied  to  these  the  name  "humic 
acid."  Berzelius  (1838)  evidently  had  the  general  meaning  of  the 
term  "humus"  in  mind  when  he  used  the  term  "humin"  in  describ- 
ing certain  dark  colored  constituents  of  vegetable  mold.  Follow- 
ing the  use  of  the  term  "humin"  as  applied  to  what  was  considered 
to  be  a  definite  organic  body,  a  number  of  other  workers  took  up 
the  study  of  similar  substances,  and  a  number  of  other  terms  more 
or  less  related,  soon  appeared.  The  name  of  Mulder  (1849)  is  asso- 
ciated for  the  most  part  with  the  terms  applied  to  humus-like  sub- 
stances which  have  appeared  more  or  less  in  the  literature  from  that 
time  to  the  present.  For  instance,  he  says : 

At  present  seven  different  organic  substances  are  known  to  exist  in  the 
soil.  They  are  crenic  acid,  apocrenic  acid,  geic  acid,  humic  acid,  and  humin, 
nlmic  acid  and  ulmin. 

These  bodies  were  divided  by  him  into  two  groups,  one  con- 
sisting of  crenic  and  apocrenic  acids,  and  the  other  group  embrac- 
ing all  the  others.  According  to  Mulder  (1849)  these  seven  or- 
ganic bodies  were  intimately  related,  and  five  at  least  were  five  suc- 
cessive steps  in  the  decay  of  organic  matter  in  the  soil.  The  first 
step  in  this  decay  he  regarded  as  ulmic  acid ;  this  on  further 
oxidation  yielded  humic  acid ;  and  this  in  its  turn,  on  still  further 
oxidation,  geic  acid.  Continued  oxidation  produced  apocrenic, 
and  finally  crenic  acid. 

*(De  Candolle  1833,  p.  279)  states  "Das  Ulmin  ist  von  Klaproth  entdeckt 
ivorben." 

**  I"  have  been  unable  to  verify  the  original  citation,  which,  according  to 
Uoper  was  Kastner's  Archiv.  Bd.  7,  p.  163;  Bd.  8,  p.  145.  Presumably  one  of 
these  articles  is  that  referred  to  by  Russell  (1912)  entitled  "Ueber  Pflanzen- 
Xaturlehre,  Niirnberg,  1826. 


10 

A  number  of  chemists  have  given  the  percentage  composition 
of  the  supposed  acids,  but  no  two  agree.  Nothing  is  known  in  re- 
gard to  their  constitution.  The  lack  of  definite  chemical  character- 
ization of  these  compounds  is  stated  by  Cameron  and  Bell  (1905) 
as  follows : 

The  existence  itself  of  these  acids  has  never  been  satisfactorily  demon- 
strated. *  *  *  *  No  satisfactory  description  of  the  physical  or  chemical 
properties  of  these  supposed  acids,  their  salts,  or  characteristic  derivatives, 
have  been  recorded. 

Nearly  every  writer  on  soils  from  the  time  of  Mulder  to  Hil- 
gard  (1906,  p.  126)  spoke  of  these  acids  with  the  same  assurance 
as  of  oxalic  or  tartaric  acids,  or  any  other  organic  compound  that 
has  well  known  derivatives. 

The  early  investigators,  including  Mulder,  soon  found  that 
sugar,  starch,  carbohydrates  generally,  and  even  proteins,  when 
treated  with  strong  acids  or  alkalies,  gave  rise  to  dark  colored 
compounds  having  the  same  general  appearance  and  properties 
as  the  humus  substances  arising  in  the  soil  through  decay.  How- 
ever, a  conspicuous  feature  of  the  work  on  these  humus  substances 
is  the  discordant  results  for  preparations  bearing  the  same  name 
and  often  from  the  same  source. 

Robertson,  Irvine,  and  Dobson  (1907)  reached  conclusions  that 
although  there  were  many  strong  resemblances  between  natural 
and  artificial  humus  preparations,  in  regard  to  properties  and  com- 
position, yet  there  are  important  differences  in  constitution.  Re- 
cently Gortner  (1916  c)  has  shown  that  in  all  probability  the  humin 
formed  from  carbohydrates  is  actually  formed  by  a  polymerization 
of  furfural  which  is  in  turn  formed  from  the  carbohydrates  by  the 
action  of  the  acid.  Gortner  and  Blish  (1915),  Gortner  (1916  c),  and 
Gortner  and  Holm  (1917)  have  likewise  shown  that  the  dark  col- 
ored products  originating  in  an  acid  hydrolysis  of  protein  sub- 
stances have  their  origin  in  the  tryptophane  nucleus.  Obviously, 
if  carbohydrate  humin  originates  from  furfural,  and  protein  humins 
originate  in  the  tryptophane  nucleus,  mixtures  of  protein  and 
carbohydrate  would  produce  a  great  variety  of  physically  similar 
but  chemically  different  mixtures. 

One  of  the  important  points  at  issue  between  the  early  in- 
vestigators was  whether  humic  acid  and  allied  bodies  contained 
nitrogen  as  a  constituent.  Mulder  (1849,  1862)  held  that  nitrogen 
was  not  a  constituent  of  these  substances,  but  was  present  as 
ammonia,  that  is  the  acids  were  present  in  the  soil  as  ammonium 
salts,  and  in  this  connection  he  says : 

In  good  arable  soil — that  is,  one  in  which  the  organic  constituents  are  as 
far  as  possible  decomposed — none  of  these  substances  contain  nitrogen 
as  a  constituent  element;  all  their  nitrogen  exists  in  the  state  of  ammonia. 

Detmer  (1871)  came  to  the  opposite  conclusion,  claiming  that 
nitrogen  in  humic  acid  as  usually  obtained,  was  present  in  organic 
combination  (not,  however,  bound  in  the  humic  acid  molecule). 
He  obtained  his  humus  by  digesting  with  alkali.  After  precipitat- 
ing with  acid,  he  redissolved  it  in  ammonia  and  precipitated  the 
mineral  constituents  with  phosphoric  and  oxalic  acids  and  am- 
monium sulphide.  After  treating  with  potassium  hydroxide  and 
precipitating  with  hydrochloric  acid,  he  obtained  a  preparation 


11 

containing  1.5  per  cent  nitrogen.  No  ammonia  was  evolved  on 
making  alkaline,  but  about  23  per  cent  of  the  total  nitrogen  was 
evolved  on  treatment  with  sodium  hypobromite.  Detmer  believed, 
however,  that  humic  acid  did  not  contain  nitrogen,  but  that  the 
nitrogen  found  was  present  in  some  organic  compound  occurring  as 
an  impurity  in  his  humic  acid  preparation.  By  a  tedious  process 
he  was  able  to  lower  the  nitrogen  content  of  his  humic  acid  to 
0.179  per  cent. 

Ritthausen  (1877)  attributed  the  high  nitrogen  content  of  peat 
to  the  formation  of  complex,  difficultly  decomposable  materials  by 
absorption  of  ammonia  and  pointed  to  the  low  ammonia  content  as 
an  indication  of  it,  claiming  it  was  not  present  as  such  after  absorp- 
tion. In  an  attempt  to  disprove  Ritthausen's  theory,  Sivers  (1880) 
found  that  he  could  expel  only  very  small  amounts  of  ammonia  by 
heating  with  potassium  hydroxide.  He  concluded  that  all  the  am- 
monia taken  in  remained  as  such,  and  did  not  go  to  form  complex 
compounds.  He  maintained  that  most  of  the  nitrogen  was  in  the 
form  of  protein  but  presented  no  conclusive  evidence.  Grouven 
(1883)  tried  to  show  that  the  nitrogen  of  humus  was  due  to  the 
absorption  of  ammonia  by  humic  acids,  but  found  frojn  various 
samples  that  only  one-fiftieth  of  the  total  nitrogen  was  liberated 
on  heating  with  milk  of  lime,  and  only  one-twentieth  when  heated 
for  two  hours  with  potassium  hydroxide. 

It  was  found  by  Loges  (1886)  that  the  hydrochloric  acid  ex- 
tract of  the  soil  gave  a  precipitate  with  phosphotungstic  acid,  which 
is  recognized  as  being  a  precipitating  agent  for  certain  nitrogenous 
compounds.  Baumann  (1887)  found  that  certain  black  Russian 
soils  rich  in  humus,  containing  but  small  traces  of  ammonia  in  the 
soil,  gave  a  considerable  amount  of  it  on  boiling  the  soil  with  dilute 
hydrochloric  acid.  From  this  he  suggested  the  presence  of  amino 
and  amide  compounds  in  the  soil.  About  the  same  time  this  sub- 
ject was  more  thoroughly  investigated  by  Berthelot  and  Andre 
(1886).  They  found  that  the  nitrogenous  matter  was  split  up  pro- 
ducing ammonia  and  soluble  nitrogenous  compounds,  and  that  the 
hydrolysis  goes  further  the  greater  the  strength  of  the  acid,  the 
longer  it  is  in  contact  with  the  soil  and  the  higher  the  temperature. 
A  soil  containing  0.174  per  cent  of  nitrogen  was  heated  on  a  water 
bath  for  two  hours  with  7  per  cent  hydrochloric  acid  and  31.9  per 
cent  of  its  nitrogen  was  dissolved.  Of  this  soluble  nitrogen  17.8 
per  cent  was  ammonia.  Similar  experiments  were  conducted  using 
3.4  per  cent  hydrochloric  acid,  and  0.7  per  cent  hydrochloric  acid 
and  distilled  water. 

Warington  (1887)  working  with  a  sample  of  Rothamsted  soil, 
which  had  been  heavily  manured,  showed  the  presence  of  a  small 
amount  of  amide  nitrogen  by  using  both  hypobromite  and  nitrous 
acid.  It  seems  highly  probable  from  these  experiments  that  at 
least  a  part  of  the  nitrogen  in  the  soil  is  present  as  amino  com- 
pounds. Eggertz  (1888)  found  that  the  nitrogen  content  of  thir- 
teen samples  of  humus  varied  from  2.59  to  6.43  per  cent  and  states 
that  the  nitrogen  was  present  in  organic  form  and  not  as  an  am- 
monium salt.  Sestini  (1899)  also  showed  the  presence  of  amino 


12 

nitrogen  by  the  action  of  nitrous  acid.  Dojarenko  (1902)  working 
with  humus  from  seven  Russian  soils  found  appreciable  quantities 
of  amino  nitrogen.  Unlike  previous  investigators  he  determined 
the  amount  present  quantitatively,  and  assumed  all  of  the 
amino  nitrogen  was  present  as  amino  acids.  He  also  made  deter- 
minations of  the  ammonia  by  distillation  with  magnesium  oxide, 
and  obtained  the  amide  nitrogen  by  hydrolysis  with  dilute  hydro- 
chloric acid  and  subsequent  distillation  of  the  ammonia  formed 
with  magnesium  oxide. 

B.     The  Humus  Theory  of  Grandeau. 

A  tremendous  impetus  was  given  to  the  study  of  soils  by  the 
work  of  Grandeau,  because  he  believed  that  the  ammonia  extract 
of  soils  contained  the  nutritive  substances  essential  for  the  life 
of  the  plant  and  for  the  fertility  of  the  soil.  The  theory  that  the 
humus  extract  was  of  such  value  had  a  great  deal  to  do  with  re- 
tarding the  development  of  the  study  of  the  organic  matter  of  the 
soil. 

The  method  of  Grandeau  (1872)  is  essentially  the  one  in  use 
at  the  present  time  in  America  for  the  determination  of  humus.  He 
elaborated  a  method  for  the  estimation  of  the  "matiere  noire"  of 
the  soil  by  first  leaching  the  soil  with  dilute  acid  in  order 
to  set  the  humus  free  from  its  combination  with  the  alkaline  earths, 
removing  the  excess  of  acid  by  washing  with  water,  then  moisten- 
ing the  soil  with  ammonia  and  allowing  it  to  stand  for  a  short 
time  (three  to  four  hours,  cf.  Grandeau  1877,  p.  149),  after  which 
the  humus  solution  was  displaced  by  repeated  washings  with  am- 
moniacal  water.  The  dark  brown  solution  so  obtained  was  evapor- 
ated to  dryness  in  platinum,  weighed,  ashed,  and  the  amount  of 
"matiere  noire"  and  of  ash  recorded.  Grandeau  regarded  the  humus 
ash  as  an  integral  part  of  the  humus.  He  believed  that  the  organic 
matter  which  dissolved  was  responsible  for  the  fertility  of  the  soil, 
apparently  not  so  much  because  of  the  carbon  and  nitrogen  con- 
tent, as  for  the  high  percentage  of  phosphoric  acid  and  potash  in 
the  humus  ash. 

The  views  of  Grandeau  wrere  never  generally  adopted  in  Eu- 
rope although  often  accepted  by  individual  workers,  but  have  been 
more  generally  accredited  in  America,  due  to  the  sponsorship  of 
certain  parts  of  Grandeau's  humus  theory  by  the  late  Professor 
Hilgard.  The  American  investigators,  e.  g.,  Hilgard  (1906),  Ladd 
(1898),  and  Snyder  (1895,  1897,  and  1901),  however,  do  not  report 
the  humus  ash  as  an  integral  part  of  the  humus  but  call  only  the 
volatile  portion  humus.  They  consider  that  the  humus  is,  in  part 
at  least,  combined  in  the  soil  with  inorganic  substances ;  these 
compounds  are  called  "humates"  and  to  their  abundance  and  pro- 
duction has  been  ascribed  an  important  part  of  the  maintenance 
of  soil  fertility. 

Hilgard  (1906)  regarded  the  humus  of  the  soil  as  a  definite  soil 
product,  formed  from  vegetable  material  in  the  soil  under  the  in- 
fluence of  fungus  and  bacterial  growths ;  this  conversion  being 


13 

most  efficiently  carried  out  in  the  presence  of  only  a  moderate 
amount  of  moisture,  under  the  influence  of  a  more  or  less  rapid 
circulation  of  air  and  in  the  presence  of  calcium  carbonate  to 
neutralize  any  acids  which  may  be  formed.  Under  these  conditions 
the  vegetable  substance  is  converted  into  black,  neutral,  insoluble 
humus  compounds. 

He  believed  that  the  nitrogen  of  plant  debris  which  has  become 
an  integral  part  of  the  soil  must  first  be  converted  by  humifying 
bacteria  and  fungi  into  humus  before  the  nitrogen  can  become  avail- 
able to  the  nitrifying  bacteria  and  thus  rendered  available  for  the 
use  of  the  higher  plants.  His  views  of  the  persistence  of  plant  ma- 
terials in  soils  are  contained  in  the  following  statement: 

As  a  matter  of  course,  the  several  organic  compounds  contained  in  plants 
may  continue  to  exist  in  soils  for  some  time,  varying  according  to  conditions 
of  temperature  and  moisture.  Thus  dextrin,  glucose,  and  even  lecithin  and 
nuclein  have  been  reported  to  be  found.  The  activity  of  the  numerous  fungus 
and  bacterial  ferments  under  favoring  conditions,  will  of  course,  limit  the 
continued  existence  of  such  compounds  somewhat  narrowly  so  that  they  can 
hardly  be  considered  as  active  soil  ingredients  save  in  so  far  as  they  favor 
the  development  of  bacterial  flora. 

Suzuki  (1906-08  a)  made  a  study  of  the  formation  of  humus  by 
treating  oak  leaves  with  a  humus  soil  and  various  inorganic  com- 
pounds and  concluded  that  not  only  calcium  carbonate  but  also 
magnesium  carbonate  promoted  the  decomposition  of  moist  oak 
leaves  by  fungi,  judging  from  the  amounts  of  carbon  dioxide 
evolved.  He  further  states  that  the  opinion  of  Hilgard  corresponds 
closely  to  the  natural  conditions  of  humification.  Further  studies 
of  Suzuki  (1906-08  b)  indicate  that  protein,  starch,  and  pentosans 
contribute  to  the  formation  of  the  black  matter  of  humus,  but 
neither  fat  nor  cellulose,  and  that  protection  from  air  is  essential. 

One  of  the  latest  additions  to  the  idea  of  specific  humificatio.il 
of  plant  materials  in  the  soil  is  that  of.  Trusov  (1915).  He  inoculat- 
ed various  types  of  organic  compounds  with  soil  bacteria  for  vari- 
ous lengths  of  time  and  concludes  that  humus  has  its  origin  in 
lignin,  albumen,  starch,  chlorophyll,  and  tannic  substances;  while 
cellulose,  hemicelluloses,  mono-  and  disaccharides,  glucosides,  or- 
ganic acids  including  amino  acids,  and  wax  forming  substances 
do  not  appear  to  have  any  part  in  its  formation.  He  also  finds  that 
the  organic  nitrogenous  compounds  used  as  nutrients  for  the  micro- 
organisms may  serve  as  an  indirect  source  of  humus. 

Weir  (1915)  has  recently  questioned  the  idea  that  the  soluble 
humus  of  the  soil  is  an  indication  of  the  fertility  of  that  soil  and 
that  the  humus  nitrogen  plays  an  important  role  in  the  nutrition 
of  plants.  He  removed  40  per  cent  of  the  nitrogen  of  the  soil  by 
extracting  the  humus  with  sodium  hydroxide  and  then  used  the 
extracted  soil  for  pot  experiments.  However,  Gortner  (1916  b) 
has  shown  that  in  all  probability  a  very  considerable  portion  of  the 
humus  nitrogen  still  remained  in  Weir's  extracted  soil,  for  he  was 
able  to  extract  90.3  per  cent  of  the  original  nitrogen  content  of 
the  soil.  This  would  indicate  that  nearly  all  of  the  soil  nitrogen 
could  be  extracted  with  sodium  hydroxide. 

Snyder  (1897)  prepared  artificial  humus  by  mixing  a  subsoil 
with  certain  organic  substances  and  allowing  these  to  remain  in  a 
moist  condition  for  one  year.  At  the  end  of  the  year  humus  was 


14 

determined  on  the  resulting-  mixture  by  extraction  with  ammonia, 
folio-wing  a  previous  leaching  with  dilute  acid,  an.  I  the  humus  so 
found  was  regarded  as  having  been  formed  in  the  soil  during  the 
preceding  year.  Unfortunately  Snyder  did  not  correct  for  am- 
monia soluble  organic  matter  in  the  mixtures  at  the  beginning  of 
the  experiment.  The  results  of  Fraps  and  Hamner  (1910)  and 
Gortner  (1917)  show  that  ammonia  dissolves  a  considerable  por- 
tion of  material  from  unchanged  organic  compounds,  so  that  the 
humus  gain  at  the  end  of  the  experiment  was  in  all  probability 
actually  a  loss  when  compared  with -the  amount  of  ammonia  soluble 
materials  at  the  beginning  of  the  experiment.  Fraps  and  Hamner 
(1910)  as  well  as  Gortner  (1917)  report  a  series  of  experiments  af- 
ter the  general  plan  adopted  by  Snyder,  with  the  exception  that  the 
ammonia  soluble  materials  were  determined  both  at  the  beginning 
and  at  the  end  of  the  experiments,  and  in  each  instance  the  am- 
monia soluble  material  was  found  to  decrease. 

The  experiments  of  Gortner  (1917)  furnish  no  evidence  that  a 
specific  "humification"  of  plant  materials  takes  place  in  the  soil 
giving  rise  to  an  increased  amount  of  "humus."  He  says: 

On  the  contrary,  all  of  the  evidence  is  directly  opposed  to  such  a  con- 
clusion, and  it  appears  altogether  probable  that  the  maximum  amount  of  am- 
monia soluble  material  is  present  in  a  soil  immediately  after  a  green  manuring 
crop  has  been  plowed  under  and  before  the  'humifying'  bacteria  or  fungi 
begin  their  work. 

Fraps  and  Hamner  (1910)  showed  that  the  humus  extract  of 
soil  must  contain  substances  from  unchanged  vegetable  materials, 
while  Gortner  (1916  a)  pointed  out  that  the  extract  must  contain 
substances  from  unchanged  plant  material,  from  bacteria  and 
protozoa. 

C.     The  Complexity  of  the  Ammonia  Soluble  Material. 

With  the  development  of  chemistry  the  idea  has  been  gradu- 
ally abandoned,  that  the  ammonia  soluble  compounds  can  contain 
the  whole  of  the  organic  matter  which  is  responsible  for  the  fer- 
tility of  the  soil.  The  work  of  the  U.  S.  Bureau  of  Soils  in  the 
isolation  of  a  large  number  of  definite  organic  compounds  from 
the  soil,  has  been  a  distinct  contribution  along  this  line. 

As  has  been  suggested  by  Gortner  (1916  a) — 

If  one  speculates  on  the  nature  of  the  soil  organic  matter,  it  becomes  obvi- 
ous that  the  variety  of  compounds  which  are  present  in  a  soil  is  limited 
only  by  those  compounds  which  were  present  in  the  plants  growing  upon 
the  soil,  plus  those  compounds  which  compose  the  bodies  of  bacteria  and 
protozoa,  plus  the  compounds  contained  in  the  soil  fungi,  plus  all  the  various 
compounds  which  may  be  formed  from  the  above  sources  by  decay,  oxidation, 
and  all  the  intricate  chemical  reactions  which  take  place  in  converting  dead 
organic  material,  either  into  living  protoplasm  on  the  one  hand,  or  into 
water,  carbon  dioxide,  and  nitrogen  on  the  other.  Undoubtedly  these  organic 
compounds  are  not  the  product  of  'humification'  but  are  derived  from  un- 
changed plant  material,  from  protozoa,  or  from  bacteria. 

A  part,  or  all,   of  these   compounds   would  be   found   in   the 
"humus"  extracted  by  Grandeau's  method — 

Inasmuch  as  the  'humus'  extract  of  soils  is  undoubtedly  a  mixture  of  organic 
compounds,  many  of  which  are  colorless  and  in  all  probability  are  extracted 
from  unchanged  plant  or  animal  materials,  and  inasmuch  as  the  soil  pigment 
present  in  this  solution  probably  rarely  exceeds  40  per  cent  of  the  'humus',  a 
determination  of  'humus',  as  ordinarily  carried  out,  appears  to  be  wholly 
without  scientific  justification.  (Gortner  1916  a.) 


15 
D.     The  Presence  of  Definite  Organic  Compounds  in  the  Soil. 

The  following  types  of  pure  organic  compounds  have  been 
isolated  from  the  soil.  In  view  of  Gortner's  statement  above  it  is 
of  interest  to  note  that  all  of  these  compounds  are  colorless,  and 
that  nearly  all  of  them  were  isolated  from  an  alkaline  humus  ex- 
tract. 

1.  Paraffin  hydrocarbons. 

Hcntriacontane,  C3iH«4.     Schreiner  and  Shorey  (1910  a,  1911  a). 

2.  Alcohols 

Argosterol*,  CseHUO.     Schreiner  and  Shorey  (1909,  1909  a) 
Phytostcrol,  C26H44O,  H2O.     Schreiner  and  Shorey  (1910  a,  1911  b) 

3.  Esters 

"Glycerides  of  fatty  acids."     Schreiner  and  Shorey  (1910  a) 
"Resin  esters."     Schreiner  and  Shorey  (1910  a) 

4.  Acids 

Oxalic  acid,  COOH-COOH.     Shorey  (1913) 
Succinic  acid,  COOH-CH.-CHr-COOH.    Shorey  (1913) 
Saccharic  acid,  COOH-(CHOH)4-COOH.     Shorey  (1913) 
Acrylic  acid,  CH2=CH-COOH.     Shorey  (1913) 
a-Crotonic  acid,  CH:1-CH  =  CH-COOH.     Walters  and  Wise  (1916). 
a     (l:ll)-Monohydroxystearic    acid,    CH,(CH,)tiCHOH  (CH2)»COOH. 

Schreiner  and  Shorey  (1910  a,  1910  b) 
Dihydroxystearic      acid,      CH3(CH2)7CHOH-CHOH(CH2)TCOOH. 

Schreiner  and  Shorey  (1908  a,  1909  a) 
Benzoic  acid,  C6H5COOH.     Shorey  (1914) 
Metaoxytoluic  acid,  OH-C6H3-CH3-COOH.     Shorey  .(1914) 
Agroceric  acid,  C2iH42O3.     Schreiner  and  Shorey  (1909  a) 
Paraffinic  acid,  C24H48O2.     Schreiner  and  Shorey  (1910  a) 
Lignoccric  acid,  C:uH4*O2.     Schreiner  and  Shorey  (1910  a) 
"Resin  acids"  (?).     Schreiner  and  Shorey  (1910  a) 

5.  Aldehydes 

Salicylic  aldehyde,  C6H4-OH-CHO.     Shorey  (1913) 

Vanillin,   OH-C6H3(OCH3)-CHO.     m-methoxy-o-hydroxy  benzal- 

dehyde.     Shorey  (1914) 
Trithiobenzaldehyde,  (C6H5CSH)3.     Shorey  (1913) 

6.  Carbohydrates 

Mannitol,  CH,OH-(CHOH)4-CH2OH.     Shorey  (1913) 
Rhamnose,  CH3-(CHOH)4-CHO,  H2O.    Shorey  (1913) 
Pentosan**,  C5H8O4.     Schreiner  and  Shorey   (1910  a),  Shorey  and 
Lathrop  (1910) 

7.  Pyrimidine  derivatives 

Cytosine,    C4H5N3O.      2-oxy,    6-amino    pyrimidine.      Schreiner    and 
Shorey  (1910  a,  1910  c) 

8.  Purine  bases 

Xanthine,    CsH4N4O2.      2,    6-dioxy    purine.      Schreiner    and    Shorey 

(1910  a,  1910  c) 
Hypoxanthine,    C5H4N4O.      6-oxy    purine.      Schreiner    and    Shorey 

(1910  a,  1910  c) 

Adenine,  C5H5N5.    6-amino  purine.    Shorey  (1913) 
Guanine***,    CsHtNtO.      2-amino,    6-oxypurine.      Lathrop     (1912). 

Schreiner  and  Lathrop  (1912). 
Tetracarbonimid****,  C4H4N4O4.     Shorey  and  Walters   (1914) 

9.  Pyridine  derivatives 

a-Picoline.    -y-carboxylic    acid,    CrHrNOa.      Shorey     (1906),    Schreiner 
and  Shorey  (1908  b) 


*The  composition  of  the  compound  was  determined  by  a  single  analysis, 
made  with  0.1500  gram  of  the  substance.  It  seems  highly  improbable  that 
the  composition  could  be  obtained  accurately  from  this  amount  of  material. 

**Pentose  sugars  have  been  separated  as  hydrolysis  products  of  substances 
isolated  from  the  soil  (Schreiner  and  Shorey  1910  a). 

**This   compound   was   isolated   from  a  steam  heated  soil. 

****  This  has  been  shown  to  be  cyanuric  acid   (Wise  and  Walters  1917). 


16 

10.  Amines 

Trimethylamine,  (CH3)3N.    Shorey  (1913) 

Choline,  HON(CH3)3-CH2-CH2OH.  Trimethyl  oxyethyl  am- 
monium hydroxide.  Shorey  (1913) 

Creatinine,  C^NaO.  ^,-imino  (n)  methyl  a-keto  tetrahydro  gly- 
oxalin.  Shorey  (1911),  Schreiner,  Shorey,  Sullivan  and  Skinner 
(1911) 

11.  Organic  phosphorus  compounds 

Nucleic  acid.     Shorey  (1911  a,  1912,  and  1913) 
Lecithin,  Aso  (1904),  Stoklasa  (1911). 

12.  Amino  acids 

Arginine,          CoHu&Os.         a-amino,        g-guanidine       valerianic      acid. 

Schreiner  and  Shorey  (1910  a,  1910  d) 
Histidine,  GiHflN3O2.  a-amino,  #-imidazole  propionic  acid. 

Schreiner  and  Shorey  (1910  a,  1910  d) 
Lysine,  CeHnN3O2.  a  £-di-amino,  caproic  acid.  Shorey  (1913) 

Whether  all  of  these  compounds  have  actually  been  isolated 
is  perhaps  an  open  question,  in  view  of  the  minute  quantities  which 
were  obtained,  insufficient  in  many  cases  for  an, exact  chemical  an- 
alysis as  stated  by  Schreiner  and  Lathrop  (1911)  : 

The  amount  of  a  substance  obtained  may  be  so  small  that  extreme  puri-' 
fication  is  out  of  the  question,  and  therefore)  in  such  cases,  where  distinct 
crystalline  form  or  characteristic  tests  are  not  available  the  identification 
becomes  uncertain,  as  neither  melting  point  nor  analysis  can  be  made. 

E.     The  Origin  of  Organic  Compounds  in  the  Soil. 

Chardet  (1914)  gives  a  discussion  of  the  possible  origin  of 
certain  organic  nitrogenous  compounds  that  have  been  isolated 
from  the  soil  or  might  be  expected  to  exist. 

A  very  complete  summary  of  our  present  knowledge  of  the  or- 
ganic matter  of  the  soil  is  presented  by  Jodidi  (1914)  under  these 
headings:  I.  Introduction;  II.  The  sulphur  compounds  of  the 
soil;  III.  The  influence  of  certain  factors  on  the  quantity  of  nitro- 
gen contained  in  the  soil ;  IV.  The  nature  of  humus  substances  ac- 
cording to  the  older  authors ;  V.  The  observations  of  later  authors 
concerning  the  nature  and  behavior  of  humus  to  certain  reagents ; 
VI.  Genetic  relationship  between  the  chemical  compounds  in  the 
soil  and  those  in  plants  and  animals ;  VII.  The  nature  of  nitrogen 
compounds  in  the  soil;  VIII.  The  organic  nitrogenous  compounds 
of  the  soil ;  IX.  Separation  of  the  nitrogenous  compounds  in  a 
sulfuric  acid  extract  (i.  e.,  hydrolysate)  of  the  soil;  X.  Cleavage 
products  of  nucleoproteins ;  XL  Lecithin  products  in  the  soil;  XII. 
Pyridine  derivatives  in  the  soil ;  XIII.  Ammonification  of  amino 
acids  and  acid  amides  in  the  soil ;  XIV.  The  occurrence  of  hydro- 
carbons, alcohols,  and  aldehydes  in  the  soil;  and  XV.  The  organic 
acids  occurring  in  the  soil. 

Different  investigators  have  succeeded  in  isolating  from  soils 
the  following  nitrogenous  compounds  which  may  be  related  to 
or  derived  from  the  proteins:  tetracarbonimid,  a-picoline  y-car- 
boxylic  acid,  trimethylamine,  nucleic  acid,  arginine,  histidine, 
lysine,  proteoses,  and  peptones.  Potter  and  Snyder  (1915  b)  have 
shown  that  in  some  soils,  at  least,  free  amino  acids  and  peptides 
occur  but  the  amounts  are  very  small. 

Since  there  have  been  so  many  nitrogenous  compounds  isolat- 


17 

ed  from  the  soil  that  are  related  to  proteins,  it  is  well  to  discuss 
some  of  the  complex  organic  compounds  such  as  nucleoproteins, 
nucleic  acids,  and  lecithins  that  find  their  way  to  the  soil  through 
plant  and  animal  remains.  While  the  final  decomposition  products 
are  undoubtedly  simple  compounds  or  elements  such  as  carbon 
dioxide,  methane,  ammonia,  nitrogen,  and  hydrogen,  these  products 
are  reached  by  fairly  definite  and  well  defined  methods  of  cleav- 
age. The  process  may  be  a  rapid  one,  a  slow  one,  or  one  entirely 
arrested  at  certain  stages,  all  depending  on  the  factors  present 
in  the  soil. 

1.  Nucleoprotein  decomposition.  The  nucleoproteins  are  with- 
out doubt  the  most  complex  compounds  that  enter  the  soil.  They 
are  common  constituents  of  plants,  animals,  bacteria,  and  molds,  and 
hence  occur  wherever  these  live  or  die.  The  chemical  changes 
through  which  these  compounds  go  during  decomposition  may  be 
rendered  clear  by  the  following  scheme  presented  by  Lilienfeld 
(1892): 

Nucleoprotein 


Protein  Nuclein 

(Histone)  I 


!  I 

Protein  Nucleic  acid 

The  products  are  then  protein  and  nucleic  acid,  the  latter  of 
which  has  been  isolated  from  the  soil  (Shorey  1911  a,  1912,  and 
1913). 

2.  Nucleic  acid  decomposition.  Nucleic  acids  are  constituents 
of  all  nuclei  and  on  decomposition  yield  a  variety  of  compounds 
composed  of  carbon,  hydrogen,  oxygen,  nitrogen,  and  phosphorus. 
The  acids  occur  in  both  plant  and  animal  cells. 

Jones  (1914)  states  that  all  plant  nucleic  acids  contain  a  pen- 
tose  group,  while  on  the  other  hand  all  animal  nucleic  acids  yield 
levulinic  acid,  which  is  formed  from  a  hexose  group  in  their  mole- 
cule. 

The  hydrolysis  products  may  be  classified,  according  to  Forbes 
and  Keith  (1914)  as  follows: 

Nucleic    acids 


Phosphoric  acid  Carbohydrates  Bases 


Pentoses 
Hexoses 
Unidentified  Purine  Pyrimidine 


Guanine  .    Cytosine 
Adenine  Thymine 

Xanthine  Uracil 

Hypoxanthine 


18 

The  purine  and  also  the  pyrimidine  derivatives  can  be  changed 
one  into  the  other  by  chemical  means.  In  a  parallel  manner  much 
the  same  results  can  be  obtained  through  the  biochemical  changes 
brought  about  by  bacteria  and  enzymes.  By  chemical  agents  Kos- 
sel  and  Steudel  ( 1903)  transformed  cytosine  into  uracil.  In  like 
manner  Fischer  (1882)  changed  guanine  into  xanthine,  and  Kossel 
(1886)  changed  adenine  into  hypoxanthine.  It  was  shown  by  Schit- 
tenhelm  and  Schroter  (1904)  that  putrefactive  bacteria,  especially 
those  of  the  coli  group,  were  able  to  convert  guanine  and  adenine 
into  xanthine  and  hypoxanthine  and  that  bacteria  also  have  the 
ability  of  breaking  down  nucleic  acid  itself.  This  change  of  nucleic 
acid  is  also  accomplished  by  certain  enzymes,  the  nucleases  (cf. 
Jones  1914  and  Euler  1912). 

It  will  be  clear  from  the  above  that  the  decomposition  of 
nucleic  acid  may  take  place  in  many  steps  and  that  the  intermediate 
as  w'ell  as  the  final  products  may  be  transformed  one  into  the  other. 
This  may  be  accomplished  in  the  soil  through  the  agency  of  micro- 
organisms or  enzymes.  Schreiner  and  Lathrop  (1912)  working 
with  steam  heated  soils  found  that  from  the  heated  samples  less 
nucleic  acid  was  obtained  than  from  the  unheated  samples.  On  the 
other  hand  the  decomposition  products  of  nucleic  acid  were  present 
in  larger  amounts  in  the  heated  soil,  indicating  that  hydrolysis  of 
nucleic  acid  has  been  accomplished  in  this  manner.  That  nucleic 
acid  decomposition  does  take  place  in  the  soil  is  evidenced  by 
the  isolation  of  certain  of  its  decomposition  products,  e.  g.,  cytosine, 
xanthine,  hypoxanthine  (Schreiner  and  Shorey  1910  a,  1910  c), 
adenine  (Shorey  1913),  and  guanine  (Lathrop  1912,  Schreiner  and 
Lathrop  1912). 

3.  Lecithin  decomposition.  A  similar  instance  of  a  single 
substance  decomposing  into  several  substances  is  that  of  the 
lecithins.  They  are  closely  related  to  the  fats  in  constitution  and 
are  possible  primary  constituents  of  all  plant  and  animal  cells. 
Lecithins  are  esters  of  glycerol  with  two  molecules  of  higher  fatty 
acids  (palmitic,  stearic,  oleic  acids  or  other  unidentified  saturated 
or  unsaturated  acids)  with  a  molecule  of  phosphoric  acid,  which 
is  at  the  same  time  combined  with  the  base  choline.  Mathews 
(1915)  states  that  in  some  cases  choline  can  be  replaced  by  neurine. 
There  are  a  number  of  different  lecithins  which  are  characterized 
by  the  nature  of  the  organic  acid  radicals  present.  The  hydrolysis 
may  be  indicated  as  follows : 

L,ecithin 


Acids  Bases  Glyceryl-phosphoric  acid 

I  I 

Palmitic  Choline  Glycerol 

Stearic  or  Phosphoric  acid 

Oleic  Neurine 

or 

Other   higher 
fatty  acids. 


19 

The  base  choline  is  also  widely  distributed  in  both  plant  and 
animal  tissues  as  well  as  being  a  decomposition  product  of  lecithins. 
It  has  been  shown  to  exist  in  the  soil.  Choline  yields  neurine  by  bac- 
terial decomposition,  and  both  of  these  compounds  break  up  into 
trimethylamine.  This  substance  may  also  be  added  to  the  soil  from 
other  sources,  both  animal  and  vegetable.  As  noted  above  Stoklasa 
(1911)  obtained  evidences  of  lecithin  in  the  soil  and  Aso  (1904)  re- 
ports small  quantities  of  lecithin  present  in  soils  rich  in  organic  mat- 
ter. Choline  has  been  isolated  from  soil  (Shorey  1913).  The  tri- 
methylamine reported  by  Shorey  (1913)  possibly  had  its  origin 
in  the  lecithin  molecule  and  it  may  be  that  the  dihydroxy-stearic 
acid  of  Schreiner  and  Shorey  (1908  a,  1909  a)  and  the  mono- 
hydroxystearic  acid  (Schreiner  and  Shorey  1908  a,  1909  a)  had 
the  same  origin.  (For  a  discussion  of  the  organic  phosphorus  of 
the  soil  sec  (iortner  and  Shaw  1917). 

F.     Bacterial  Processes  Which  Influence  the  Form  of  Soil  Nitrogen. 

We  know  that  the  decomposition  of  protein  substances  can  be 
brought  about  through  bacterial  activity  or  by  the  agency  of  en- 
zymes widely  distributed  in  the  vegetable  kingdom.  We  should 
expect  any  protein  materials  present  in  the  soil  to  be  subject  to 
the  action  of  the  above  agencies.  Viewed  in  the  light  of  the 
researches  of  Emil  Fischer  (1899-06)  protein  hydrolysis  leads  to 
disruption  of  the  complex  molecule  and  the  formation  of  simple 
molecules,  as  represented  in  the  following  scheme* — 

i'  Di-amino  acids 
Mon-amino  acids 
Acid  amides 

Fischer  has  shown  that  the  amino  acid  combination  in  the  pro- 
tein molecule  may  be  represented  as  follows: 

HO  H  H  O 

I    I!  !     !    !J 

H.N-C-C N-C-C-O  H 

I  I 

R  R 

O  H 

The  group          II     !         being  known  as  the  "peptid  group."   The 

— -C-N  — 

nitrogen  in  this  group  is  in  the  form  of  the  imino  (-NH)  radical. 
Upon  hydrolysis  each  "peptid  group"  takes  up  a  molecule  of  water 
forming  a  free  carboxyl  (-COOH)  group  changing  the  imino  group 
into  an  amino  (— NH2)  group. 

As  the  protein  hydrolysis  continues,  the  proportion  of  nitrogen 
in  the  amino  form  increases  until  it  reaches  a  maximum  at  com- 
plete hydrolysis.  It  has  been  shown  by  Van  Slyke  (1910,  1911) 
that  the  amount  of  amino  nitrogen  formed  is  a  measure  rbf  the 
hydrolysis  of  the  protein  substance.  The  amino  acids  derived  from 
protein  degradation  may  be  acted  upon  by  the  bacteria  in  the  soil 
and  bring  about  chemical  changes  which  depend  largely  on  the 
character  of  the  organisms  present. 


20 

1.  Deamination  or  reduction  plays  an  important  part  in  the 
formation   of  ammonium   salts  in   the   soil.     The   amino   group   is 
.split  off  as  ammonia  and   non-nitrogenous   organic   acids   remain. 
It  is  not  certain  whether  this   process   involves   oxidation   of  the 
amino  acid  to  the  ketonic  acid  first,  or  whether  the  deamination  is 
brought  about  by  hydrolysis.     If  the  hydroxy  acids  are  first  formed 
they  are  subsequently  reduced  so  that  the  fatty  acids  are  formed 
from  the  amino  acids.     This  can  be  illustrated  by  the   following 
examples  : 

Aspartic  acid  will  give  succinic  acid,  and  this  by  loss  of  carbon 
dioxide  gives  propionic  acid. 

Tyrosine—  >p-Hydroxy-phenyl-propionic  acid—  >p-Hydroxy-phenyl-acetic 
acid—  >p-Cresol—  >  Phenol 

This   deamination   or  reduction   is   in   all   probability   what   is 
termed  in  soil  chemistry  ammonification. 

2.  Decarboxylation  or  amine  formation  involves  the  splitting 
off  of  carbon  dioxide  by  the  action  of  so-called  carboxylase  bac- 
teria.   This  may  happen  either  before  or  after  deamination.     Their 
formation  is  illustrated  in  the  following  reactions: 

CH3-CH(NH,)-COOH-        >CH:r-CH,.-NH,.+  CO2 
Alanine  Ethyl  amine 

C,iH4(OH)-CH2-CH(NH2)-COOH    -    ->CoH4(OH)-CH2-CHo-NH2  +CO2 
Tyrosine  p-Hydroxy-phenyl-ethyl  amine 


>NH2-CH2-CH:!-CH2-CH;-NH2+CO2 
Lysine  Cadaverine 

NH2-C(NH)-NHCH2-(CH2)r-CH(NH2)-COOH- 

Arginine  NH2-C(NH)-NH-CH2-(CH2)2-CH2-NH2+CO2 

Agmatine 

Tryptophane  gives  rise  to  indole  ethyl  amine. 
Mathews  (1915)  states— 

If  the  splitting:  off  of  carbon  dioxide  occurs  after  the  deamidization  an  amine 
cannot,  of  course,  be  formed,  but  the  next  lower  carboxylic  acid  is  produced 
by  way  of  the  aldehyde. 

Thus  from  tyrosine  there  may  first  be  formed  p-hydroxy- 
phenyl-pyruvic  acid,  which  may  be  reduced  to  p-hydroxy-phenyl- 
lactic  acid,  reabsorbed  and  reexcreted  in  the  urine  ;  or  the  p-hy- 
droxy-phenyl-pyruvic  acid  may  be  split  into  p-hydroxy-phenyl 
acetaldehyde  and  carbon  dioxide,  and  the  former  be  oxidized  into 
p-hydroxy-phenyl-acetic  acid,  which  is  excreted  in  the  urine. 

It  is  well  to  remember  that  any  bacteria  of  the  coli  group  will 
split  off  carbon  dioxide  from  an  amino  acid.  It  is  evident  that 
mixed  cultures  of  bacteria  may  be  present  in  the  soil  and  thus  cause 
more  than  one  type  of  splitting  to  take  place. 

3.  Hydrolysis  takes  place  with  liberation  of  carbon  dioxide 
Ehrlich  (1911)  has  shown  that  yeasts  can  convert  amino  acids  into 
alcohols,  liberating  carbon  dioxide  and  ammonia. 

C6H4(OH)-CH2-CH(NH2)-COOH  +  H2O  --  > 

Tyrosine  CoH4(OH)-CH2-C 

Tyrosol 


21 

4.  Oxidation  results  with  liberation  of  carbon  dioxide  and 
ammonia  and  the  formation  of  a  fatty  acid  containing  one  less  car- 
bon atom.  A  type  reaction  may  be  represented  as  follows : 

R-CHr-CH(NH2)-COOH  +  Oa  -      ->R-CH2-COOH  +  CO2+NH3 

That  oxidation  is  a  factor  in  the  organic  matter  of  the  soil 
is  self-evident  from  the  fact  that  carbon  dioxide  is  constantly  pres- 
ent in  the  soil  atmosphere  in  excess  of  the  amount  present  in  the 
air,  thus  representing  degradation  of  the  organic  matter  to  carbon 
dioxide  and  water,  arid  also  from  the  fact  that  ammonia  is  trans- 
formed into  nitrates,  a  process  known  in  soil  chemistry  as  nitrifica- 
tion, a  reaction  which  is  carried  out  in  the  laboratory  by  the  most 
violent  chemical  oxidation,  e.  g.,  chromic  acid.  A  further  step  in 
this  oxidation  carries  the  nitrates  through  denitrification  which  re- 
sults in  the  liberation  of  free  nitrogen. 

In  carrying  to  completion  these  processes  on  protein  material 
one  can  easily  postulate  an  almost  unlimited  number  of  organic 
compounds,  which  are  theoretically  (and  in  all  probability)  pos- 
sible. Very  recently  Robbins  (1916)  has  produced  some  evidence 
that  the  existence  of  certain  of  the  organic  compounds  in  the  soil  is 
limited  somewhat  narrowly  by  specific  bacteria,  which  either  utilize 
the  nitrogen  or  the  carbon  of  the  compound  as  a  source  of  energy. 
Thus  pyridine  is  destroyed  by  a  specific  bacterium  which  is  able  to 
utilize  the  nitrogen,  and  the  carbon  of  cumarin  and  vanillin  is  like- 
wise a  source  of  carbon  for  other  specific  bacteria. 

G.     Nitrogen  Distribution  in  the  Soil 

The  chemistry  of  soil  nitrogen  may  to  a  large  extent  be  con- 
sidered as  being  the  chemistry  of  protein  undergoing  hydrolysis. 
The  isolation  of  a  number  of  amino  acids  indicates  that  proteins  are 
decomposed  in  the  soil  in  much  the  same  way  as  in  acid  hydrolysis 
or  animal  digestion.  Just  how  far  the  cleavages  have  already  gone 
in  the  soil  previous  to  acid  hydrolysis  remains  a  matter  of  much 
work  before  definite  conclusions  can  be  drawn. 

Walters  (1915)  has  reported  the  presence  of  certain  decompo- 
sition products  in  the  soil,  presumably  proteoses  and  peptones, 
resulting  from  either  a  partial  hydrolysis  of  proteins  or  by  the  syn- 
thetic action  of  microorganisms.  It  has  been  recorded  by  Hoppe- 
Seyler  (1909,  p.  413)  that  intermediate  protein  decomposition  prod- 
ucts may  result  from  the  action  of  water  at  high  temperature,  by 
mineral  acids,  alkalies,  oxidizing  agents,  enzymes  and  microorgan- 
isms. There  is  little  reason  to  suppose  that  the  action  of  micro- 
organisms is  other  than  that  of  the  enzymes  which  they  produce. 
Effront  (1914)  states  that  under  the  influence  of  the  various  tryp- 
sins  secreted  by  putrefactive  bacteria,  the  protein  molecule  is  split 
into  proteoses,  peptones  and  amino  acids.  The  proteoses  and  pep- 
tones represent  stages  of  decomposition  between  that  of  true  pro- 
teins and  amino  acids.  Walters  concludes — 

that  proteins  undergo  hydrolytic  decomposition  in  the  soil  in   much   the  same 
way  as  in  dig-estion  by  enzymes,  acids,  or  alkalies,   in  the  laboratory. 


22 

In  an  extensive  examination  of  the  nitrogen  compounds  of 
processed  fertilizers,  Lathrop  (1914)  has  reported  the  presence  of 
certain  protein-like  substances  similar  to  those  described  above. 

In  his  studies  on  the  chemical  nature  of  the  organic  nitrogen 
in  the  soil,  Jodidi  (1911)  thought  water  would  be  preferable  to 
either  acids  or  alkalies  for  the  purpose  of  extraction,  since  it  would 
not  be  so  liable  to  alter  the  organic  nitrogenous  materials.  He 
found  that  the  direct  extraction  of  a  soil  by  boiling  with  water  for 
ten  hours  removed  only  2.92  per  cent,  and  for  twenty-four  hours 
the  highest  amount  removed  from  any  soil  was  9.96  per  cent  of  the 
total  soil  nitrogen.  Shmook  (1914),  however,  reports  19.10  per 
cent  of  the  total  nitrogen  of  a  Laterite  soil  of  Russia  to  be  water 
soluble. 

The  literature  has  been  very  thoroughly  summarized  by  Potter 
and  Snyder  (1914)  in  regard  to  the  determination  of  ammonia  in 
soils.  Both  their  work  and  that  of  Jodidi  ( 1909)  indicates  that  the 
amount  of  ammonia  is  small.  Kelley  and  Thompson  (1914)  in  a 
study  of  some  Hawaiian  soils  reached  the  conclusion  that  ammonia 
and  nitrate  nitrogen  constitute  but  a  small  percentage  of  the  total 
nitrogen,  and  that  the  nitrogen  is  very  largely  in  organic  combina- 
tion. 

It  is  known  that  only  a  small  part  of  the  soil  nitrogen  is  dis- 
solved by  dilute  acids,  yet  it  has  been  shown  by  Kelley  and  Thomp- 
son (1914)  that  1  per  cent  hydrochloric  acid  dissolves  some  organ- 
ic nitrogen,  for  in  every  instance  the  soils  contained  only  about 
half  as  much  ammonia  nitrogen  as  was  extracted  by  the  acid. 

In  the  soil  studies  of  Potter  and  Snyder  (1915  a)  they  find  that 
the  nitrogen  extracted  by  1  per  cent  hydrochloric  acid  varied  from 
about -1.2  to  2.3  per  cent  of  the  total  nitrogen,  except  in  the  case  of 
the  peat  it  was  only  0.67  per  cent.  This  is  contrary  to  the  findings 
of  Gortncr  (1916  a).  Working  with  eight  mineral  soils  he  finds  a 
maximum  of  4.18  per  cent  of  the  total  nitrogen  soluble  in  1  per 
cent  hydrochloric  acid  with  an  average  of  3.17  per  cent.  In  three 
peats  he  finds  a  maximum  of  7.50  per  cent  with  an  average  of  3.78 
per  cent,  and  in  five  samples  of  unchanged  vegetable  materials  (oat 
straw,  alfalfa  hay,  oak  leaves,  sweet  fern  leaves,  and  grass  from  a 
peat  bog)  he  finds  a  maximum  of  34.58  per  cent  with  an  average  of 
20.10  per  cent.  These  findings  would  seem  to  indicate  that  in  the 
transformation  of  vegetable  materials  into  the  true  organic  mat- 
ter of  the  soil  there  is  a  fall  in  the  proportion  of  the  total  nitrogen 
soluble  in  very  dilute  acids. 

Shorey  (1905)  published  results  of  his  investigations  which 
gave  the  first  definite  knowledge  of  the  individual  amino  acids 
formed  in  the  decomposition  of  soil  organic  matter.  He  worked  on 
a  Hawaiian  soil  with  a  view  to  classifying  the  decomposition  prod- 
ucts of  the  nitrogenous  substances  in  the  soil.  The  method  applied 
was  that  proposed  by  Osborne  and  Harris  (1903)  for  classifying  the 
decomposition  products  of  proteins  resulting  from  acid  hydrolysis. 
The  method  is  a  modification  of  that  proposed  by  Hausmann  (1899) 
and  is  in  short  as  follows :  After  hydrolysis  the  excess  of  the 


23 

mineral  acid  is  removed  by  evaporation,  and  the  nitrogen  present 
as  ammonia  determined  by  distilling  with  an  excess  of  magnesium 
oxide ;  after  separating  the  magnesia  precipitate  from  the  remain- 
ing solution  by  filtration,  the  nitrogen  was  determined  in  the  pre- 
cipitate by  the  Kjcldahl  method,  the  di-amino  nitrogen  in  the  fil- 
trate was  precipitated  by  phosphotungstic  acid  and  determined  by 
the  method  of  Kjeldahl  and  the  mon-amino  nitrogen  determined 
by  difference. 

He  obtained  in  the  acid  solution  84.5  per  cent  of  the  total  nitro- 
gen in  the  soil,  52.3  per  cent  of  which  was  found  in  the  magnesia 
precipitate.  This  result  is  in  striking  contrast  to  those  obtained  by 
Osborne  and  Harris  (1903)  working  on  pure  proteins,  where  they 
found  that  the  nitrogen  contained  in  the  magnesia  precipitate  does 
not  usually  exceed  4  per  cent  of  the  total  nitrogen  and  in  most 
cases  is  very  much  less.  The  amount  of  nitrogen  insoluble  in  the 
12  per  cent  acids  used  in  the  digestion  may  be  designated  as  "hu- 
min." The  nitrogen  in  the  magnesia  precipitate  has  been  desig- 
nated by  most  investigators  "humin"  nitrogen.  The  total  "humin" 
nitrogen  in  the  soil  is  then  represented  by  the  nitrogen  in  the 
magnesia  precipitate  plus  that  retained  by  the  soil.  On  -recalcula- 
tion of  his  data  it  was  found  that  the  insoluble  humin  in  the  soil 
after  hydrolysis  amounted  to  15.3  per  cent  of  the  total  nitrogen, 
making  a  total  humin  nitrogen  content  of  59.1  per  cent.  This  very 
high  result  of  total  humin  nitrogen  was  undoubtedly  due  to  the  soil 
being  hydrolyzed  only  seven  hours  with  a  relatively  low  concen- 
tration of  hydrochloric  acid  and  the  insoluble  residue  boiled  the 
same  length  of  time  with  sulfuric  acid.  Complete  decomposi- 
tion of  the  proteins  probably  did  not  take  place  in  the  dilute  acids 
used  in  the  short  time  that  they  were  heated.  As  a  result  partially 
hydrolyzed  residues  may  have  been  precipitated  by  the  magnesium 
oxide,  which  would  account  for  the  high  results. 

Shorey  (1906)  concluded  that  even  though  we  might  know 
much  concerning  the  constitution  of  the  compounds  comprising 
the  various  groups  isolated  from  protein  by  this  method  of  analysis, 
we  know  nothing  concerning  their  composition  when  isolated  from 
soil,  inasmuch  as  we  are  not  dealing  with  a  pure  protein  (cf.  also 
Oiortner  1913,  1914,  1916  c). 

The  work  of  Suzuki  (1906-08  c)  gives  us  further  knowledge  of 
the  individual  amino  compounds  formed  in  the  decomposition  of  soil 
organic  matter.  He  worked  with  three  samples  of  humic  acid,  o>ne 
obtained  from  Merck  (origin  unknown  to  Suzuki),  one  prepared 
from  an  unmanured  soil,  and  one  from  a  compost  heap.  After 
boiling  each  preparation  for  ten  hours  with  strong  hydrochloric 
acid,  the  undecomposed  residue  was  filtered  off,  washed,  and  the 
residue  extracted  twice  in  this  manner  with  strong  hydrochloric 
acid.  He  determined  the  amounts  dissolved  as  amide,  di-amino,  and 
mon-amino  acid  nitrogen.  From  65  to  75  per  cent  of  the  total 
nitrogen  was  dissolved  by  the  hydrochloric  acid  and  in  the  extract 
41  to  62  per  cent  of  the  nitrogen  was  not  precipitated  by  phospho- 
tungstic acid.  A  sample  of  humic  acid  was  twice  extracted,  with 


24 

concentrated  acid  and  the  residue  analyzed.  His  results  calculated 
on  the  ash  free  basis  showed  the  residue  to  contain  64.11  per  cent 
carbon,  3.35  per  cent  hydrogen,  and  0.80  per  cent  nitrogen.  The 
residue  becomes  lower  in  nitrogen*,  hydrogen,  and  ash  but  richer 
in  carbon  as  the  hydrolysis  is  continued. 

Detmer  (1871)  pointed  out  that  similar  results  were  true  in 
peat  beds  where  the  deposits  remained  undisturbed  for  years.  He 
found  that  there  is  an  increasing  carbon  and  nitrogen  content  of 
the  humus  for  varying  depths.  This  is  shown  by  the  following 
table : 


Carbon 

Hydrogen 

Oxygen 

Nitrogen 

Brown  peat,  near  the  surface... 
Dark  peat,  7  feet  

57.75 
62.02 

5.43 
521 

36.02 
3067 

0.80 
2  10 

Black  peat,  14  feet.  

64.07 

5.01 

26.87 

4.05 

Likewise    Gortner    (1917)    observed — 

that  there  is  a  much  greater  wastage  of  carbon  than  nitrogen.  Hilgard 
(1906)  calls  attention  to  the  increased  nitrogen  content  of  the  humus  over 
that  of  the  original  vegetable  materials.  If  we  take  the  average  carbon  con- 
tent of  proteins  as  51.15  per  cent  (average  of  30  analyses  given  by  Mathews 
1915)  a  C:N  ratio  of  3.06  found  in  soil  A-1916  would  give  a  nitrogen  content 
of  16.71  per  cent,  which  approaches  very  nearly  to  the  average  nitrogen  content 
of  these  30  proteins,  i.  e.,  17.66  per  cent.  It  is  evident  that  the  materials  re- 
maining in  the  soils  are  rapidly  increasing  in  nitrogen  content. 

Suzuki  (1906-08  c)  made  further  studies  on  a  500  gram  sample 
of  the  humic  acid  obtained  from  Merck.  It  was  hydrolyzed  with 
concentrated  acid  and  the  solution  obtained  subjected  to  esterifica- 
tion  and  fractional  distillation  according  to  the  method  of  Fischer 
(1901).  He  obtained: 

Alanine    • 2.39  gm. 

Leucine    , 2.16  gm. 

Alanine  +  aminovalerianic  acid  • 0.11  gm. 

Aminovalerianic  acid 0.57  gm. 

Proline   (copper  salt  of  active  proline)  .  • 0.67  gm. 

(copper  salt  of  inactive  proline)  (?) 0.50  gm. 

Aspartic  acid • 0.06  gm. 

Impure  aspartic  acid   (?) •  •   3.16  gm. 

Glutaminic   acid    present 

Tyrosine    trace 

Histidine    trace 

Ammonia    1.90  gm. 

Copper  salts  of  unknown  acids •  • • •  • 30.30  gm. 

As  these  compounds  are  typical  protein  decomposition  prod- 
ucts, his  work  proves  that  the  humic  acid  examined  by  him  was 
either  of  a  protein  nature,  a  mixture  of  protein  decomposition  prod- 
ucts, or  probably  both  together  with  some  as  yet  unknown  com- 
pounds. Unfortunately,  the  origin  of  the  acid  was  unknown  to 
Suzuki,  but  he  states  that  it  was  probably  prepared  from  peat. 

From  a  study  on  Michigan  peat  soils  Jodidi  (1909)  has  con- 
cluded that  the  bulk  of  the  organic  nitrogen  is  made  up  of  acid 
amides,  di-amino  acids,  and  mon-amino  acids.  He  used  slightly 
modified  methods.  The  ammonia  was  determined  as  above  by 
distillation  with  magnesium  oxide.  The  residue  from  distillation 
with  magnesia  was  dissolved  in  dilute  sulfuric  acid  and  the 

*He  stated  that  although  the  nitrogen  content  decreases,  it  is  very  difficult 
to  entirely  remove  all  of  the  nitrogen. 


25 

di-amino  acids  precipitated  by  phosphotungstic  acid.  The  nitrogen 
in  the  precipitate  of  di-amino  acids  was  determined  by  the  method 
of  Kjeldahl.  The  filtrate  from  the  di-amino  acids  containing  the 
mon-amino  acids  was  oxidized  by  the  Kjeldahl  method  and  the 
nitrogen  determined.  In  some  cases  he  secured  the  mon-amino 
nitrogen  by  difference,  stating  that  it  was  difficult  to  get  a  direct 
determination  of  the  mon-amino  nitrogen  by  the  Kjeldahl  method. 
He  states  that— 

this   percentage   was   usually   higher   than   the   one   directly   found   by   kjeldahl- 
izing   the   filtrate   from    the    phosphotung-istic   acid   precipitate. 

In  one  experiment  the  percentage  of  mon-amino  nitrogen  by 
direct  determination  was  62.83  per  cent,  while  by  difference  the 
result  was  67.22,  and  in  another  case  the  results  were  64.25  and 
65.06  respectively. 

It  will  be  noted  that  this  is  a  departure  from  the  method  used 
by  Shorey  (1905)  in  that  here  the  nitrogen  is  separated  into  three 
fractions  instead  of  the  usual  four.v  The  nitrogen  in  the  magnesia 
precipitate  was  distributed  with  the  di-amino  and  mon-amino  acid 
nitrogen.  This  method  of  nitrogen  distribution  will  be  classed 
as  "Jodidi  numbers"  (in  contrast  to  the  Hausmann  numbers)  in 
the  subsequent  portion  of  the  paper. 

Van  Slyke's  (1910)  nitrous  acid  method  was  first  applied  by 
Robinson  (1911)  to  a  study  of  peat  soil,  in  order  to  determine  the 
amount  of  amino^nitrogen  present.  The  ammonia  nitrogen  was  re- 
moved by  previous  distillation  with  magnesium  oxide.  The  only 
value  of  Robinson's  work  seems  to  be  in  the  fact  that  his  figures 
for  total  and  amino  nitrogen  increase  to  a  maximum  with  in- 
creasing time  of  hydrolysis,  in  much  the  same  manner  that  pro- 
teins react ;  thus  indicating  that  the  amino  groups  were  not  ex- 
isting free  in  the  peat  but  in  some  form  of  combination  which 
did  not  react  with  nitrous  acid.  For  example,  after  one  hour's 
hydrolysis  the  total  nitrogen  of  the  soil  in  solution  amounted  to 
29.86  per  cent,  while  the  amino  nitrogen  was  4.62  per  cent  or  a 
ratio  exceeding  6:1.  After  forty-two  hours'  hydrolysis  the  nitro- 
gen of  the  soil  in  solution  was  51.54  per  cent  of  the  total  nitrogen 
and  the  amino  nitrogen  was  25.07  or  a  ratio  only  slightly  exceed- 
ing 2:1.  This  ratio  increases  again  with  further  hydrolysis  so 
that  at  the  end  of  138  hours  the  ratio  is  almost  3:1.  However,  the 
amount  of  nitrogen  in  solution  was  so  small  that  the  experimental 
error  of  measuring  total  and  amino  nitrogen  must  have  been 
quite  large. 

More  recently  Jodidi  (1911)  made  a  study  of  some  Iowa  soils 
using  a  modification  of  the  Osborne  and  Harris  (1903)  method. 
The  nitrogen  removed  from  the  solution  by  the  magnesium  oxide 
was  ignored  by  the  author.*  This  contained  a  part  of  the  so-called 
"htimin"  nitrogen.  Subtracting  the  sum  of  the  ammonia**  and 
di-amino  nitrogen  from  100  he  found  the  per  cent  of  the  nitrogen  in 

^Experimental  data  presented  later  in  this  paper  will  show  that  this 
fraction  may  exceed  9  per  cent  of  the  total  nitrogen. 

**He  distinguishes  the  ammonia  nitrogen  originally  present  in  the  soil 
as  such,  from  that  produced  by  acid  hydrolysis. 


26 

solution  as  mon-amino  nitrogen.  It  will  be  readily  seen  this  con- 
clusion is  incorrect.  The  mon-amino  acid  nitrogen  as  deter- 
mined represents  the  sum  of  the  humin  and  mon-amino  nitrogen. 
It  is  very  unfortunate  that  this  mistake  should  have  been  made 
since  this  gives  us  only  the  actual  ammonia  and  di-amino  acid 
nitrogen  for  use  in  comparison  with  other  investigations  of  the 
organic  soil  nitrogen  as  distributed  by  acid  hydrolysis.  Kelley 
(1914)  following  details  as  outlined  by  Jodidi  (1911)  has  made 
the  same  error  and  the  criticisms  above  apply  with  equal  force  to 
his  data. 

It  is  also  extremely  unfortunate  that  investigators  should  in 
any  case  rely  upon  figures  obtained  "by  difference"  for  any  one  of 
their  fractions.  It  is  sometimes  permissible  to  use  figures  obtained 
in  this  manner,  for  example,  in  case  a  determination  has  been  lost 
and  lack  of  time  or  other  consideration  prevents  a  repetition  ; 
but  to  constantly  use  figures  obtained  in  this  manner  is  unscientific, 
especially,  since  by  this  method  we  have  no  means  of  determining 
how  great  the  experimental  error  of  the  method  may  have  been. 

Jodidi  (1911)  called  attention  to  the  fact  that  in  the  case  of 
protein  substances  the  distillation  of  the  hydrolyzed  protein  with 
magnesium  oxide  gives  pure  ammonia.  This,  however,  may  not 
hold  true  for  the  hydrolyzed  portion  of  soils,  since  some  protein 
substances  through  decay  yield  organic  bases.  It  has  been  shown 
by  Bocklisch  (1885)  that  dimethyl  amine  is  formed  through  putre- 
faction of  fish,  and  trimethylamine  has  been  produced  by  the 
putrefaction  of  wheat  flour  and  fish.  The  bases  putrescine  and 
cadaverinc  result  from  the  decay  of  organic  substances  under  cer- 
tain conditions.  It  is  possible  for  certain  di-amino  acids  to  be- 
come transformed  into  di-amines,  as  for  example,  arginine  can  be 
decomposed  into  urea  and  ornithine  through  bacterial  activity. 
These  processes  can  be  expressed  by  the  following  equations  : 

NH2-C(NH)-NH-CH2-(CH2)2-CH(NH2)-COOH  +  H20- 

Arginine 

NH2 


Hs-CHr-CH^CH  (NH:)-COOH+C=O 

l 

Ornithine  .NH, 

Urea 
NH,,-CH2-CH,-CHa-CH(NH2)-COOH  --  > 

Ornithine 

CO2+NH^CH,-CH2-CH,-CH2-NH2 
PUtrescine 

Jodidi  found  that  the  ammonia  obtained  by  distilling  the 
evaporated  extract  of  the  soil  with  magnesium  oxide  was  actually 
pure  ammonia,  thereby  establishing  the  absence  of  any  volatile 
organic  bases  ;  but  that  the  phosphotungstic  acid  precipitate  and  the 
filtrate  from  that  precipitate  did  not  represent  di-amino  and  mon- 
amino  acids  only. 

In  order  to  find  out  how  much  of  the  di-amino  and  mon-amino 
nitrogen  actually  belonged  to  di-amino  and  mon-amino  acids,  the 
solutions  were  subjected  to  analysis  by  the  formaldehyde-titration 


27 

method  of  Schiff  (1900,  1901,  1902)  as  modified  by  Sorensen  (1908), 
Henriques  (1909),  and  Henriques  and  Sorensen  (1910). 

In  a  comparison  of  the  amount  of  di-amino*  acid  nitrogen, 
calculated  as  if  histidine,  arginine,  and  lysine  were  present  in  about 
equal  amounts,  he  finds  that  in  plot  E,  101.8  per  cent;  plot  Q,  84.8 
per  cent;  and  plot  U,  93.9  per  cent  of  the  nitrogen  in  the  phos- 
photungstate  fraction  was  actually  present  as  di-amino  acids. 

However,  he  obtains  widely  divergent  results  for  mon-amino 
acid  nitrogen;  in  plot  E,  91.64  per  cent;  plot  Q,  52.63  per  cent; 
plot  U,  40.12  per  cent;  plot  H,  88.31  per  cent;  and  plot  J,  92.11 
per  cent  of  the  total  nitrogen  in  the  "filtrate  from  the  bases"  was 
actually  present  as  mon-amino  acid  nitrogen  whereas  all  should 
have  been  present  in  this  form  if  dealing  with  pure  proteins  only. 
He,  therefore,  concludes  that  the  di-amino  and  mon-amino  acids, 
or  in  other  words,  the  bases  and  filtrate  from  the  bases  by  hydro- 
lyzing  soils,  contain  other  products- than  are  formed  by  hydrolysis 
of  pure  proteins. 

In  a  series  of  fertilized  soils  studied  by  Lathrop  an.d  Brown 
(1911)  they  find  that  almost  98  per  cent  of  the  nitrogen  in  the  soil 
is  of  organic  nature.  The  ammonia  and  nitrate  nitrogen  constitute 
the  remainder.  Employing  the  same  method  to  the  distribution  of 
the  soil  organic  nitrogen  as  Shorey  (1905),  they  boiled  100  grams  of 
soil  with  500  cc.  of  hydrochloric  acid  (sp.  gr.  1.115)  for  three  hours, 
and  used  the  filtrate  after  making  to  definite  volume  for  the 
analyses.  The  figures  given  for  ammonia  nitrogen  represent  the 
actual  amount  of  nitrogen  as  ammonia  obtained  by  hydrolysis  and 
does  not  include  the  ammonia  nitrogen  already  present  in  the 
soil.  They  find  that  the  plots  which  have  received  organic  fer- 
tilizers give  the  largest  amount  of  ammonia  on  hydrolysis,  the 
amount  being  highest  in  the  plot  which  has  received  manure  alone 
and  lowest  in  the  check  plot. 

Of  the  five  soils  studied,  four  contained  over  25  per  cent  of 
the  "humin"  nitrogen  soluble  in  acid,  while  the  other  only  showed 
about  half  as  much.  Since  the  nitrogen  of  the  soil  not  soluble  in 
acid  may  be  considered  "humin"  nitrogen,  the  total  amount  in  this 
form  in  the  above  four  soils  was  over  53  per  cent,  while  in  the 
other  soil,  which  received  dried  blood,  it  amounted  to  only  43  per 
cent. 

However,  the  fractions  which  they  determined  have  actually 
very  little  significance  in  a  discussion  of  protein  hydrolysis  products, 
inasmuch  as  a  three-hour  hydrolysis  is  far  too  short  a  time  to  com- 
pletely decompose  the  protein  molecule.  This  explains  their  high 
figures  for  humin  nitrogen  and  low  ones  for  mon-amino  acid 
nitrogen.  The  di-amino  and  mon-amino  acid  nitrogen  differ  rather 
widely  but  there  seems  to  be  no  agreement  between  the  form  of 
nitrogen  and  the  plot  treatment. 


*The    exact    interpretation    of   his    data    is    difficult    to    understand. 


28 


In    conclusion    they    say — 


these  five  samples  of  soil  are  really  the  same  soil  under  long-  continued 
treatment  of  different  kinds.  It  is  not  improbable  that  work  on  widely  differ- 
ent soils  will  show  even  much  greater  variations  than  those  here  noted. 
The  work  shows,  however,  that  even  in  such  cases  there  is  a  difference  in 
the  nitrogenous  compounds  in  the  soil,  and  that  different  decompositions  of 
the  nitrogenous  matter  has  taken  place  and  probably  will  continue  to  take 
place,  under  the  different  conditions  imposed  upon  the  soils  in  the  field. 

A  very  interesting  study  has  been  made  by  Shmook  (1914) 
of  the  nitrogen  distribution  in  four  Russian  soils,  one  of  the  Podzol 
type,  two  of  the  Chernozem  type,  and  one  of  the  Laterite  type  by 
applying  the  method  of  Hausmann  (1899).  The  water  extract 
from  100  grams  of  the  Podzol  soil  showed  a  very  high  content  of 
soluble  nitrogenous  compounds.  This  amounted  to  0.0452  gram 
of  nitrogen  which  constituted  19.10  per  cent  of  the  total  soil  nitro- 
gen. This  was  distributed  as  follows :  amide  nitrogen  0.0034 
gram,  di-amino  nitrogen  traces,  and  mon-amino  nitrog-eia  0.0408 
gram.  These  results  were  deducted  from  the  analyses  of  the 
hydrolyzed  soil.  Thirty  gram  samples  of  soil  were  hydrolyzed 
with  120  cc.  of  hydrochloric  acid  (sp.  gr.  1.12)  for  8  hours  and 
the  analysis  carried  out  as  directed. 

He  finds,  that  the  Chernozem  and  Podzol  soils  show  a  simi- 
larity in  the  distribution  of  amide  nitrogen,  and  that  of  the  amino 
acid  nitrogen,  but  that  the  nitrogen  distribution  in  the  Laterite 
soil  is  entirely  different  from  the  other  types.  He  concludes  that 
the  amount  of  protein  in  the  soil  is  not  in  direct  relation  to  the 
amount  of  organic  matter,  and  that  the  nitrogen  insoluble  in  hydro- 
chloric acid  occurs  in  unknown  form  and  composes  only  1.50  to 
1.90  per  cent  of  the  organic  matter  of  the  Chernozem  and  Podzol 
soils,  but  13.70  per  cent  of  the  total  organic  matter  of  the  Laterite 
soil,  after  subtraction  of  the  protein  nitrogen  belonging  to  this 
insoluble  portion.  He  suggests  that  these  results  would  indicate 
that  the  organic  nitrogen  existed  in  the  soil  in  large  part  as  protein 
material  in  the  Chernozem  and  Podzol  soils,  but  that  a  considerable 
portion  was  of  a  non-protein  origin  in  the  Laterite  soil,  since 
the  amount  of  this  insoluble  "melanin"  in  pure  proteins  amounts 
to  from  0.60  to  1.80  per  cent  of  the  total  protein  nitrogen.* 

Potter  and  Snyder  (1915  a)  made  a  study  of  some  Iowa  soils 
using  Van  Slyke's  (1911)  method  of  protein  analysis.  Their  soils 
were  the  same  type  but  had  received  different  fertilizer  treatment. 
At  the  same  time  they  also  made  a  study  of  peat  soil.  The  soils 
were  in  all  cases  first  extracted  with  1  per  cent  hydrochloric  acid 
"in  order  to  render  the  humus  more  soluble."  They  were  then 
hydrolyzed  by  boiling  one  part  of  the  soil  with  two  parts  of  22 
per  cent  hydrochloric  acid  for  forty-eight  hours.  They  also  pre- 
pared a  1  per  cent  sodium  hydroxide  extract  of  the  acid  leached 
soils,  and  after  precipitation  with  sulfuric  and  acetic  acids  the 
resulting  humic  acid  precipitate  was  subjected  to  the  above  method 
of  analysis. 

The  authors  conclude:  (1)  that  the  humin  nitrogen  as  deter- 
mined by  the  Van  Slyke  method  on  soils  extracted  by  dilute  alkali 

*Actually  in  some  cas^s  these  results  are  much  lower,  and  in  others  de- 
cidedly Higher,  e.  gr.,  Van  Slyke  (1911)  finds  gelatin  contains  0.07  per  cent  and 
fibrin  3.17  per  cent. 


29 

is  very  high  when  compared  to  the  amounts  in  proteins ;  (2)  that 
no  typical  class  of  organic  compounds  is  extracted  from  the  soil 
by  dilute  alkali ;  (3)  that  the  amounts  of  amino  acid  and  peptid 
nitrogen  in  the  soil  are  found  to  be  very  small  compared  to  the 
amounts  of  amino.  acids  formed  by  acid  hydrolysis ;  (4)  (and  this 
is  the  most  important  for  our  purpose)  that 

nothing  very  significant  can  be  deduced  from  the  variations  in  the  different 
soils, 

or  in  other  words,  the  organic  matter  in  the  same  soil  type  under 
different  fertilizer  treatment,  is  essentially  the  same,  and  as  I  shall 
show  later  in  the  experimental  part  of  this  paper,  the  organic  mat- 
ter (as  distributed  by  Van  Slyke's  method)  in  different  soil  types  is 
essentially  the  same. 

Lathrop  (1916)  recently  made  a  study  of  protein  decomposi- 
tion in  the  soil.  He  added  a  high  grade  nitrogenous  fertilizer  to 
the  soil  and  allowed  decomposition  to  proceed  at  laboratory  tem- 
perature, and  at  different  periods  topk  samples  and  subjected  them 
to  Van  Slyke's  method  o.f  protein  analysis  in  order  to  determine 
how  the  different  fractions  were  affected  by  bacteria  and  other 
agencies  present  in  the  soil. 

From  his  \vork  he  concludes  that  the  analysis  obtained  by  the 
Van  Slyke  method  indicates  that  there  is  a  formation  of  protein 
taking  place  in  the  soil  in  the  course  of  the  decomposition  of  the 
protein  materials,  and  that  apparently  the  new  protein  is  somewhat 
resistant  to  decomposition.  He  states,  that — 

this  is  indicated  in  (1)  the  unequal  loss  of  mon-amino  acids  and  hydroly/able 
nitrogen  from  the  soil  during-  the  early  stages,  (2)  by  an  increase  in  amide 
nitrogen  during  the  early  stages,  (3)  by  an  increase  in  histidine  nitrogen  dur- 
ing the  early  stages,  (4)  by  an  increase  in  the  arginine  nitrogen  during  the 
later  stages,  and  (5)  by  an  increase  in  lysine  nitrogen  during  the  later  stages. 

This  view  that  the  protein  nitrogen  in  the  soil  was  largely  con- 
tained in  the  bodies  of  bacteria  and  protozoa  had  been  previously 
advanced  by  Shmook  (1914). 

It  was  stated  by  Loew  and  Aso  (1906-08)  that  under  favor- 
able condition  of  growth  protein  material  is  excreted  by  yeast  and 
bacteria,  and  that  soluble  materials  can  pass  through  the  cytoplasm 
to  the  outside  on  death  of  the  cell.  They  also  state  that  the  amount 
of  nitrogenous  substances  partly  consisting  of  peptones  excreted 
by  dead  cells,  is  by  no  means  inconsiderable. 

H.     A  Summary  of  the  Nature  of  the  Organic  Matter  of  the  Soil  in 
the  Light  of  Our  Present  Knowledge. 

It  has  been  pointed  out,  that  the  organic  matter  of  the  soil 
was  at  first  considered  to  be  a  very  simple  thing,  that  the  alkali 
extract  contained  the  essential  plant  nutrients,  and  that  the  process 
of  "humification"  was  the  necessary  step  through  which  the  or- 
ganic matter  of  the  oil  must  pass  in  order  to  be  converted  into  food 
materials  for  plant  life ;  but  as  the  knowledge  of  chemistry  de- 
veloped it  became  evident  that  the  problem  was  more  complex. 
A  definite  knowledge  of  the  forms  of  organic  matter  in  the  soil 
can  only  be  secured  when  we  have  a  thorough  understanding  of 


30 

the  chemical  products  formed  by  the  action  of  the  bacteria,  pro- 
tozoa, and  fungi  on  each  other,  and  on  the  organic  matter,  both 
animal  and  plant,  that  finds  its  way  to  the  soil.  The  present  method 
is  to  study  the  soil  with  its  complex  mixture  of  organic  and  in- 
organic compounds,  and  by  the  application  of  recognized  methods 
of  chemical  investigation  unravel  the  mysteries  tied  up  in  it; 
but  unless  we  have  the  climatic  and  cultural  conditions  uniform, 
we  cannot  hope  to  secure  results  that  will  be  of  general  application. 

There  have  been  two  methods  of  attacking  the  problem:  first, 
the  isolation  and  study  of  the  individual  organic  compounds  ex- 
isting in  the  soil,  and,  second,  a  study  of  the  hydrolysis  products. 
It  is  evident  that  these  methods  are  slow  and  tedious  and  unsatis- 
factory, but  on  the  other  hand  a  study  of  all  the  possible  combina- 
tions of  the  organic  substances  existing  in  the  soil  appears  to  be 
an  endless  task  in  the  light  of  our  present  knowledge.  In  brief, 
a  complete  and  thorough  knowledge  of  the  organic  matter  of  the 
soil  appears  possible  only  after  we  have  mastered  all  of  the  bio- 
chemical processes  which  are  characteristic  of  the  fungi,  protozoa, 
and  bacteria  of  the  soil,  and  have  a  much  deeper  insight  into 
the  chemical  constitution  of  vegetable  cells  than  we  have  at  the 
present  time.  Our  present  knowledge  leads  us  to  believe  that 
it  is  possible  to  isolate  an  almost  infinite  variety  of  chemical 
compounds  from  a  soil,  the  number  and  variety  reaching  a  limit 
only  when  we  have  isolated  all  of  the  compounds  which  are  pres- 
ent in  the  plants  which  grew  upon  the  soil,  plus  those  compounds 
contained  in  the  bodies  of  bacteria,  protozoa,  and  fungi,  plus  all  of 
the  compounds  which  may  be  derived  from  these  compounds 
under  the  peculiar  soil  conditions  of  decay,  oxidation,  bacterial  ac- 
tion, and  the  secretions  of  fungi  and  living  plant  roots. 


31 


II.     EXPERIMENTAL:     A  STUDY   OF  THE  NITROGEN 
DISTRIBUTION  IN  DIFFERENT  SOIL  TYPES. 

A.  The  Problem. 

It  has  been  shown  in  the  historical  study  above  that  a  number 
of  investigators  have  studied  the  organic  nitrogen  distribution  in 
the  soil  by  applying  either  Hausmann's  (1899)  or  Van  Slyke's  (1910, 
1911)  method  of  protein  analysis.  It  has  been  demonstrated  by 
Potter  and  Snyder  that  various  plots  on  a  single  soil  type  under 
different  fertilizer  treatments  gave,  with  Van  Slyke's  method,  es- 
sentially the  same  results. 

I  have  taken  up  the  problem  at  this  point  and  made  a  similar 
study  of  the  organic  nitrogen  distribution  in  different  soil  types  in 
an  attempt  to  see  whether  the  forms  in  which  nitrogen  occur 
differ  from  locality  to  locality  and  from  soil  type  to  soil  type.  1 
am  concerned  with  the  problem  of  distribution  of  the  organic  nitro- 
gen in  the  soils  and  soil  extracts  studied. 

B.  The  Material. 

The  study  was  made  using  two  peats,  one  muck,  seven  mineral 
surface  soils,  and  one  subsoil.  All  were  from  the  State  of  Min- 
nesota. The  origin  and  type  names  are  in  accordance  with  the 
surveys  of  the  Bureau  of  Soils  of  the  U.  S.  Department  of  Agri- 
culture when  such  surveys  were  available.  Almost  all  of  the  soils 
used  are  portions  of  the  identical  samples  employed  by  Gortner 
(1916  a,  and  1917)  in  his  soil  studies.  The  samples  used  in  this 
study  were  all  air  dry  soils.  The  moisture  was  determined  by 
heating  the  soils  to  a  temperature  of  100°  C.  for  12  hours  and  then 
weighing. 

The  descriptions  of  the  soils  follow : 

1.  Calcareous  black  grass-peat.     This   sample   was   selected 
from  a   large  bulk  sample  taken  to  a  depth  of  8  inches  from   a 
grass  bog  near  the  Agricultural  Experiment  Station  Farm,  St.  Paul. 
The  peat  was  black  and  well  decomposed.     The  peat  was  ground 
to  a  powder  in  a  ball  mill  before  using.    The  air  dry  material  con- 
tained 6.40  per  cent  moisture. 

2.  Sphagnum-covered  peat.    This  sample  of  very  strongly  acid 
peat  was  prepared  from  a  large  bulk  sample  taken  from  a  bog  on 
the  Experimental  Farm  near  Grand  Rapids.     The  superficial  layer 
of  sphagnum  and  shrubs  was  first  removed  and  a  sample  of  the  un- 
derlying peat  taken  to  a  depth  of  8  inches.     The  peat  was  poorly 
decomposed.    The  sample  was  prepared  by  grinding  to<  a  powder  in 
a  ball  mill.    The  air  dry  soil  contained  5.90  per  cent  moisture. 

3.  Acid  "muck"  soil.     A  sample  of  this  strongly  acid  soil  was 
obtained  from  a  bog  about  two  miles  from  the  farm  of  the  Agri- 


32 

cultural  Experiment  Station,  St.  Paul.  The  sample  is  a  composite 
of  10  samples  taken  to  a  depth  of  8  inches  lengthwise  of  the  bog. 
The  vegetation  of  the  bog  was  largely  cattails  (Typha  spp.)  and 
rushes  (Scirpus  spp.)  The  sample  contained  5.60  per  cent  moisture 
in  the  air  dry  condition. 

4.  Fargo   clay   loam.     The    sample    analyzed    consisted    of    a 
composite  made  from  144  borings  taken  to  a  depth  of  8  inches  from 
a  twenty-acre  field  on  the  Northwest  Sub-station  Farm,  Crookston. 
The  sample  was  highly  calcareous.     This  soil  type  has  been  de- 
scribed by  Mangum  and  Schroeder  (1906).     The  moisture  content 
of  the  air  dry  soil  was  3.92  per  cent. 

5.  Fargo  silt  loam.     This  sample  was  a  composite  made  from 
100  borings  to  a  depth  of  6  inches,  twenty  borings  being  taken  from 
each  of  five  virgin  fields  near  Nerstrand,  Rice  County.     The  sam- 
ple had  a  neutral -reaction.     The  soil  type  has  been  described  by 
Burke  and  Kolbe  (1909).    The  air  dry  soil  contained  14.89  per  cent 
moisture. 

6.  Carrington  silt  loam.     This  soil  is  represented  by  two  sam- 
ples.    Each  composite  consisted  of   100  borings   to  a  depth   o-f  6 
inches,  twenty  borings  being  taken  from  each  of  five  virgin  fields 
near  Nerstrand,   Rice  County,  for  Sample  number  I,  and  twenty 
borings  being  taken  from  each  of  five  virgin  fields  near  Morristown, 
Rice  County,  for  Sample  number  II.     Sample  1  was  strongly  acid 
while  Sample  II  was  but  slightly  acid.     The  soil  type  is  described 
by  Burke  and  Kolbe  (1909).    The  moisture  content  of  sample  I  was 
6.22  per  cent. 

7.  Hempstead  silt  loam.     A  composite  sample  from  36  borings 
to  a  depth  of  6  inches  was  taken  from  12  plots  on  the  Agricultural 
Experiment  Station  Farm,  St.  Paul.     No  commercial  fertilizer  had 
been  applied,  but  the  land  had  long  been  under  cultivation.     The 
soil  type  has  been  described  by  Smith  and  Kirk  (1914).    The  sam- 
ple was  strongly  acid.     The  air  dry  soil  contained  3.07  per  cent 
moisture. 

8.  Prairie-covered  loess.     The  sample  consisted  of  50  borings 
taken  to  a  depth  of  one  foot,  ten  borings  being  taken  from  each  of 
five  virgin   fields  near   Luverne,   Rock   County.     The   sample  was 
somewhat  calcareous.     The  area  from  which  the  sample  was  ob- 
tained has  not  been  subjected  to  a  detailed  soil  survey.     The  air 
dry  soil  contained  7.89  per  cent  moisture. 

9.  Forest-covered   loess.     This   sample   wras   taken   from   five 
virgin   fields   near   Caledonia,   Houston   County,   ten   borings   to   a 
depth  of  one  foot  being  taken  from  each  field  and  equal  weights 
from  each  boring  being  combined  in  the  composite  sample.     The 
sample  was  strongly  acid.    The  air  dry  soil  had  a  moisture  content 
of   1.87  per  cent. 

10.  Hempstead  silt  loam  subsoil.     This  was  a  third  foot  bulk 
sample  taken  from  a  grove  on  the  farm  of  the  Agricultural  Experi- 
ment Station,  St.  Paul.     The  sample  was  strongly  acid. 


33 
C.     The  Method. 

The  method  of  Van  Slyke  (1911)  has  been  used  throughout 
this  investigation  because  the  nitrogen  can  be  separated  into  a 
larger  number  of  fractions  than  when  the  earlier  method  of  Haus- 
mann  (1899)  is  employed.  The  different  fractions,  however,  are 
not  listed  in  the  same  manner  as  in  the  Van  Slyke  method,  for 
since  we  are  not  dealing-  with  pure  protein  material  we  cannot 
correctly  speak  of  arginine,  histidine,  cystine,  and  lysine  nitrogen. 

Van  Slyke  (1915)  has  called  attention  to  the  fact  that  his 
method  was  devised  for  the  analysis  of  pure  protein  material  and 
not  for  a  heterogeneous  mixture  of  nitrogen  compounds.  This 
fact  was  not  recognized  by  certain  investigators.  Grindley  and 
Slater  (1915)  applied  this  method  to  the  analysis  ot  feeding  stuffs 
in  exactly  the  same  manner  as  though  they  were  dealing  with  a 
pure  protein.  Potter  and  Snydcr  (1915  a)  analyzed  certain  soils 
and  soil  extracts  and  report  their  fractions  as  "arginine,"  "histi- 
dirtfe,"  etc.  Although  they  state  (p.  2221)  : 

It  is  not  thought  that  nitrogen  as  found  by  the  Van  Slyke  method,  work- 
ing with  such  a  complex  as  the  soil,  is  in  reality,  all  lysine,  histidine,  etc., 
nitrogen.  It  might  be  said  that  each  group,  as  found,  represents 'a  class  of 
compounds  having  the  particular  reaction  by  which  the  lysine.  histidine,  etc., 
nitrogen  respectively  are  determined. 

It  is  obvious  that  there  are  other  types  of  organic  materials 
which  will  interfere  with  the  nitrogen  distribution.  It  seems  very 
probable  that  in  soils  as  well  as  in  the  material  analyzed  by  Grind- 
ley  and  Slater  (1915)  there  must  be  many  organic  nitrogen  com- 
pounds which  have  no  relation  to  the  protein  molecule,  such  as 
purme  bases,  pyrimidine  bases,  nitrogenous  lipins,  nitrogenous  pig- 
ments, as  well  as  other  non-protein  nitrogenous  compounds  (cf. 
the  list  of  non-protein  nitrogenous  compounds  actually  isolated 
from  the  soil  as  given  in  the  preceding  part  of  this  paper).  Gortner 
(1913)  states  that  much  valuable  comparative  data  can  be  obtained 
by  the  application  of  Van  Slyke's  method  to  the  analysis  of  heter- 
ogeneous materials ;  but  it  is  self  evident  that  no  analogy  can  be 
drawn  between  the  analysis  of  pure  protein  and  the  analysis  of  a 
protein  mixed  with  an  unknown  amount  of  foreign  nitrogenous 
compounds.  The  results  of  Potter  and  Snyder  (1915  a)  are  of 
little  value  in  advancing  our  knowledge  of  soil  proteins  or  for 
comparison  with  analyses  of  pure  proteins,  but  are  extremely 
valuable  and  interesting  for  comparison  between  themselves  and 
with  other  analyses  of  soils  carried  out  under  similar  conditions. 
It  must  be  remembered  that  all  data  on  similar  material  is  strictly 
comparable  when  the  same  method  of  analysis  is  followed. 

It  is  possible  that  many  of  the  non-protein  nitrogenous  com- 
pounds may  be  split  up  during  the  hydrolysis  of  heterogeneous  ma- 
terial. Gortner  (1913)  has  shown  that  uric  acid  nitrogen  is  dis- 
tributed in  all  four  of  the  major  fractions  after  hydrolysis.  The 
ammonia  nitrogen  amounted  to  15.27  per  cent,  humin  nitrogen 
35.98  per  cent,  basic  nitrogen  12.97  per  cent,  and  non-basic  nitrogen 
3S./8  per  cent.  "The  humin  nitrogen  contained  no  trace  of  black 
color  and  was  probably  calcium  ureate."  Probably  all  of  the  purines 
and  pyrimidines  would  behave  in  a  similar  manner. 


34 

The  general  method  employed  in  this  investigation  will  be  dis- 
cussed in  detail  for  two  soils,  a  peat  and  a  mineral  soil,  inasmuch  as 
the  experimental  conditions  vary  in  minor  details  with  the  two 
types. 

1.  The  method  in  detail  for  a  peat  soil.  Duplicate  samples 
of  15  grams  were  hydrolyzed  in  the  presence  of  hydrochloric  acid 
for  48  hours.  The  content  of  calcium  oxide  was  taken  into, account, 
and  corrections  made  so  that  the  100  cc.  of  hydrochloric  acid  used 
was  of  sufficient  concentration  to  neutralize  the  lime  and  at  the 
same  time  furnish  a  constant  boiling  acid.  The  hydrolysis  was 
carried  out  in  200  cc.  long  neck,  round  bottom  Kjeldahl  flasks,  fitted 
with  modified  Hopkins  condensers  made  from  a  test  tube  which 
fit  rather  loosely  into  the  neck  of  the  flask.  By  means  of  this  device 
any  error  due  to  products  extracted  from  cork  or  rubber  stoppers 
was  obviated.  The  flasks  were  heated  to  gentle  boiling  on  the 
same  sand  bath  over  an  Argand  burner,  so  that  the  rate  of  hydro- 
lysis would  be  as  near  the  same  as  possible. 

After  completion  of  the  48  hour  hydrolysis  the  mixture  was 
evaporated  in  a  Claissen  distilling  flask  under  diminished  pressure 
until  all  the  hydrochloric  acid  possible  was  driven  off.  The  residue 
after  this  distillation  was  dissolved  in  100-150  cc.  of  water,  100  cc. 
of  95  per  cent  alcohol,  and  an  excess  of  calcium  hydroxide  sus- 
pended in  water  was  added  and  the  ammonia  distilled  off  into 
standard  acid  at  a  temperature  of  40-50°  C.  under  a  pressure  of  less 
than  30  mm.,  distillation  being  continued  for  at  least  a  half  hour. 
The  results  are  listed  under  "ammonia  nitrogen." 

The  alkaline  mixture  in  the  distilling  flask  was  filtered  and 
the  precipitate  well  washed  with  hot  water  until  free  of  chlorides. 
A  Kjeldahl  determination  was  made  of  the  filter  and  its  contents, 
and  the  results  listed  under  "humin  nitrogen." 

The  filtrate  and  washings  from  the  humin  were  acidified  with 
hydrochloric  acid  and  concentrated  under  diminished  pressure  to 
less  than  200  cc.  To  this  solution  was  added  18  cc.  of  concen- 
trated hydrochloric  acid  and  the  whole  heated  on  the  water  bath 
until  hot.  A  solution  containing  15  grams  of  phosphotungstic  acid 
was  then  added  and  the  heating  on  the  water  bath  continued  for 
an  hour.  The  flask  was  then  set  aside  in  a  cool  place  for  48  hours. 
The  precipitate  of  the  bases  was  then  filtered  off  and  washed  as 
directed  by  Van  Slyke  (1911). 

The  basic  phosphotungstates  were  suspended  in  800  cc.  of 
water  and  brought  into  solution  by  the  cautious  addition  of  a  50 
per  cent  solution  of  sodium  hydroxide,  a  few  drops  of  phenolph- 
thalein  being  added  to  guard  against  too  great  an  excess  of  alkali. 
The  phophotungstic  acid  was  precipitated  by  the  addition  of  a 
slight  excess  of  20  per  cent  barium  chloride,  and  the  barium  phos- 
photungstate  was  filtered  off  and  washed  free  of  chlorides  with 
hot  water. 

The  filtrate  and  washings  were  united,  acidified  with  hydro- 
chloric acid,  and  concentrated  under  diminished  pressure  to  a  very 
small  volume.  After  cooling  the  residue  was  filtered,  washed  and 
made  up  to  50  cc.  volume. 


35 

The  washed  precipitate  of  barium  phosphotungstate  and  filter 
were  subjected  to  Kjeldahl  determination  for  any  nitrogen  that 
might  be  held  by  absorption,  adsorption,  or  occlusion,  as  was  also 
the  filter  and  contents  remaining  after  the  final  filtration  of  the 
solution  containing  the  basic  nitrogen.  In  all  cases  some  nitrogen 
was  found.  This  nitrogen  is  probably  derived  from  the  "unad- 
sorbed  humin  carried  down  with  the  !^asic  phosphotungstates" 
mentioned  by  Van  Slyke  (1915,  p.  284).  Inasmuch  as  my  work  was 
done  prior  to  this  publication,  I  added  this  nitrogen  to  the  total 
nitrogen  content  of  the  bases  instead  of  to  the  humin. 

During  the  distillation  after  kjeldahling  this  precipitate  the 
cochineal  indicator  took  on  a  color  which  made  the  acid  solution 
appear  that  complete  neutralization  might  have  occurred  when  in 
fact  it  had  not.  This  color  change  was  noticed  in  every  case  with 
the  barium  phosphotungstate  distillate.  This  made  titration  diffi- 
cult, since  a  new  end  point  had  to  be  established.  Gortner  and 
Holm  (private  communication)  have  observed  a  similar  color 
change  in  the  case  of  fibrin  hydrolyzed  in  the  presence  of  a  large 
excess  of  formaldehyde.  They  explain  this  finding  on  the  assump- 
tion that  pyridine  (or  some  similar  base)  is  formed  which  is  not 
easily  broken  down  in  the  Kjedahl  process  (cf.  Dakin  and 
Dudley,  1914),  and  which  greatly  influences  the  color  changes  of 
the  indicator  when  the  base  is  volatalized  during  the  subsequent 
distillation  with  alkali.  Whether  or  not  this  is  the  cause  of  the 
phenomenon  observed  in  my  materials  cannot  be  ascertained,  with- 
out further  investigation. 

In  no  case  did  I  attempt  to  separate  the  basic  nitrogen  into 
the  usual  fractions  of  "arginine,"  "cystine,"  "histidine,"  and  "lysine" 
nitrogen,  because  I  am  not  dealing  with  pure  protein.  Instead  in 
each  case  the  total  nitrogen  liberated  as  ammonia  was  determined 
on  25  cc.  of  the  solution  containing  the  bases.  This  was  determined 
in  exactly  the  same  manner  that  Van  Slyke  used  for  the  determina- 
tion of  arginine  nitrogen.  The  volume  of  standard  acid  neutralized 
by  the  ammonia  indicated  the  amount  contained  in  the  25  cc.  of 
solution  used.  The  nitrogen  found  is  listed  as  "basic  nitrogen  set 
free  as  ammonia  by  50  per  cent  potassium  hydroxide."  The  solution 
remaining"  from  this  determination  was  used  in  the  estimation  of 
the  total  nitrogen  of  the  bases.  This  was  performed  according  to 
Van  Slyke's  directions.  The  quantity  of  acid  neutralized  in  this 
determination  was  added  to  that  neutralized  in  the  basic-nitrogen- 
set-free-as-ammonia-by-50-per-cent-potassium-hydroxide,  thus  se- 
curing- the  "total  basic  nitrogen." 

The  "amino  nitrogen  of  the  bases""  was  determined  in  Van 
Slyke's  (1912)  apparatus,  using  10  cc.  portions  of  the  solution. 

The  filtrate  from  the  bases  was  treated  with  sodium  hydroxide 
solution  until  a  slight  turbid  precipitate -of  lime  was  formed,  and 
then  cleared  immediately  by  the  addition  of  acetic  acid.  This  was 
concentrated  under  diminished  pressure  and  on  cooling  was  made 
to  200  cc.  volume.  The  solutions  were  more  or  less  violet  in  color. 
"Total  nitrogen  in  the  filtrate  from  the  bases"  was  determined  on 
duplicate  portions  of  25  cc.  each  by  the  method  of  Kjeldahl.  The 


36 

digestion  was  continued  for  three  hours  after  the  solutions  were 
clear,  so  that  the  phosphotungstic  acid  would  not  interfere  with 
the  accuracy  of  the  determination.  The  "amino  nitrogen  in  the 
filtrate  from  the  bases"  was  determined  on  duplicate  portions  of 
10  cc.  each  by  means  of  the  Van  Slyke  (1912)  apparatus. 

2.  The  method  in  detail  for  a  mineral  soil.  Duplicate  portions 
of  250  grams  were  hydrolyzed  in  500  cc.  round  bottom  Kjeldahl 
flasks  for  48  hours  on  different  sand  baths.  Allowance  was  always 
made  for  the  lime  content  of  the  soil,  and  the  requisite  amount  of 
hydrochloric  acid  added  to  insure  the  presence  of  a  constant  boiling 
acid  (sp.  gr.  1.115),  and  a  volume  of  approximately  250  cc.  The 
solutions  boiled  smoothly  and  gave  no  trouble  by  bumping.  Air 
dry  soil  was  used  in  all  cases,  but  the  moisture  was  determined  on 
a  separate  portion  and  all  data  calculated  to  the  dry  basis. 

On  completion  of  the  hydrolysis  each  of  the  two  samples  was 
diluted  to  a  1000  cc.  in  measuring  flasks  and  allowed  to  settle  for 
at  least  24  hours.  The  clear  solution  was  then  syphoned  off  and 
an  aliquot  of  500  cc.  analyzed  according  to  the  usual  method  of 
Van  Slyke.  In  nearly  all  cases  this  solution  was  straw  color  due 
to  the  presence  of  ferric  salts  that  had  been  formed  during  the 
hydrolysis.  No  black  color,  the  usual  color  of  a  protein  hydrolysate, 
was  observed  in  any  instance. 

Another  aliquot  of  100  cc.  was  used  for  the  determination  of 
total  nitrogen  in  the  solution  by  making  duplicate  Kjeldahl  de- 
terminations on  50  cc.  portions.  A  second  aliquot  of  100  cc.  was 
used  for  the  determination  of  ujodidi  numbers"  (cf.  p.  25)  when 
they  were  determined. 

The  soil  remaining  in  the  measuring  flask  was  washed  free 
from  soluble  nitrogen  with  a  1  per  cent  solution  of  potassium  sul- 
fate by  decantation  from  tall  soil  beakers,  the  solution  after  set- 
tling being  syphoned  off  not  oftener  than  twice  a  day.  This  meth- 
od was  employed  in  order  to  prevent  the  clay  from  forming  a 
colloidal  suspension.  Distilled  water  alone  would  remove  all  elec- 
trolytes and  allow  a  portion  of  the  clay  to  remain  in  the  solution 
in  colloidal  suspension.  It  is  known  that  suspensions  of  finely  di- 
vided clay  carry  a  negative  charge  in  pure  water.  Since  it  is 
necessary  for  the  complete  precipitation  of  colloids  to.  have  some 
electrolyte  present  it  was  decided  to  use  a  1  per  cent  solution  of 
potassium  sulfate.  The  negatively  charged  colloid  was  thus  pre- 
cipitated by  the  positive  ions  of  the  potassium  sulfate  solution 
and  at  the  same  time  the  salt  would  not  interfere  with  the  subse- 
quent Kjeldahl  determination. 

A  concrete  example  of  the  thoroughness  of  this  washing  may 
well  be  given.  It  will  be  noted  that  700  cc.  of  the  original  hydrol- 
ysate was  syphoned  off  for  the  different  analyses.  This  left  a  total 
volume  of  300  cc.  of  residue  and  solution  to  be  washed  by  decanta- 
tion with  1  per  cent  potassium  sulfate.  By  the  methods  of  calcu- 
lation given  in  the  following  paragraphs  it  was  found  that  the  re- 
maining- solution  contained  0.1089  gram  of  nitrogen.  If  three- 
fourths  of  the  wash  solution  is  removed  each  time,  there  will  remain 


37 

in  the  solution  at  the  end  of  the  fourth  washing  approximately  0.0004 
gram  of  the  original  nitrogen.  Actual  Kjeldahl  determinations 
were  made  on  250  cc.  portions  from  the  fourth  washing  in  the  case 
of  duplicates  from  the  same  soil,  and  the  results  indicated  that 
0.0006  gram  of  nitrogen  still  remained  in  the  solution.  Since  this 
was  within  experimental  error  of  the  theoretical  value,  the  method 
of  washing  by  decantation  was  followed  in  all  the  subsequent  work 
with  mineral  soils,  or  those  which  had  mineral  soils  added  previous 
to  the  analysis. 

The  residue  from  the  hydrolyzed  soil  was  evaporated  to  dry- 
ness  on  the  steam  bath  in  an  evaporating  dish,  then  further  dried 
at  about  110°  C.  After  cooling,  this  dry  soil  was  passed  through  a 
1  mm.  sieve  and  after  being  thoroughly  sampled,  duplicate  nitro- 
gen determinations  were  made  on  15  gram  portions  and  the  total 
nitrogen  remaining  in  the  soil  calculated.  These  results  are  listed 
as  ''insoluble  humin  nitrogen  in  the  soil."  The  weight  of  the  dry 
soil  divided  by  the  average  specific  gravity  (2.6)  represented  the 
actual  volume  occupied  by  this  soil  residue.  The  total  volume  of 
the  hydrolysate  minus  the  volume  occupied  by  the  insoluble  resi- 
due gives  the  actual  volume  of  the  soil  solution. 

Since  the  analysis  was  made  on  500  cc.  of  the  soil  solution  it 
was  necessary  to  recalculate  the  total  "insoluble  humin  nitrogen  in 
the  soil"  in  order  to  determine  the  amount  of  this  humin  nitrogen 
actually  belonging  to  the  aliquot  analyzed. 

The  total  nitrogen  belonging  to  the  solution  analyzed  was 
found  by  taking  the  sum  of  the  total  nitrogen  in  the  solution  and 
the  above  insoluble  humin  nitrogen.  Knowing  the  total  nitrogen 
content  of  the  soil  before  hydrolysis  and  the  total  nitrogen  in  solu- 
tion, the  per  cent  of  the  total  nitrogen  in  solution  after  hydrolysis 
can  be  determined. 

The  500  cc.  aliquot  was  concentrated  under  reduced  pressure 
until  the  hydrochloric  acid  was  practically  removed  and  the  am- 
monia nitrogen  determined  in  the  manner  outlined  under  the  peat 
analysis. 

The  "humin"  fraction  precipitated  by  the  calcium  hydroxide 
was  almost  colorless  or  light  yellow,  due  to  the  iron  salts  con- 
tained in  it.  This  bulky  precipitate  was  always  washed  by  de- 
cantation after  the  method  above  described,  except  that  distilled 
water  was  used,  the  united  washings  being  concentrated  to  200 
cc.  or  less  for  the  precipitation  of  the  basic  nitrogen. 

It  was  found  necessary  to  use  35  grams  of  phosphotungstic 
acid  for  the  precipitation  of  the  bases.  The  remainder  of  the 
analysis  was  carried  out  as  directed  under  peats. 

3.  The  method  for  determination  of  "Jodidi  numbers."  A 
100  cc.  portion  of  the  clear  hydrolysate  was  concentrated  under 
reduced  pressure  and  the  ammonia  nitrogen  determined  in  the 
usual  manner.  The  residue  remaining  in  the  flask  after  this  de- 
termination was  dissolved  in  concentrated  hydrochloric  acid  and 
phosphotungstic  acid  added.*  After  standing  the  usual  length  of 
time  the  precipitate  was  filtered  of!  and  the  total  nitrogen  deter- 

*lt  will  be  noted  that  no  "humin"  fraction  is  separated.  In  that  respect 
the  "Jodidi  numbers"  differ  from  Hausmann  numbers. 


38 

mined  on  the  filter  and -contents  by  the  Kjeldahl  method  and  listed 
as  "basic  N."  The  filtrate  from  the  above  precipitate  was  con- 
centrated and  made  to  300  cc.  volume.  Duplicate  determinations 
were  made  on  100  cc.  portions,  and  the  total  nitrogen  listed  as 
nitrogen  in  the  filtrate  from  the  "bases." 

4.  The  determination  of  nitrogen.  Nitrogen  was  determined 
on  the  soils  and  soil  extracts  in  the  usual  manner,  using  25-35  cc. 
H.,SO4,  10  gm.  K2SO4,  and  a  crystal  of  CuSO4.  All  titrations  were 
made  with  N/14  acid  and  alkali  so  that  the  figures  obtained  repre- 
sented milligrams  of  nitrogen  without  necessitating  a  calculation. 

D.     The  Analytical  Data. 

The  essential  data  which  have  already  been  published  on  the 
soils  studied  are  shown  in  Table  I. 

Table  I. — Certain  analytical  data  for  the  soils  used  in  this  paper. 
Data  of  Gortncr  (1$16  a). 


3 

'c  "^ 

&  "" 

11 

^  o 

o   tf  "w 

1^ 

|1 

be 

c    G  X 

•**   in 

^    C  _o 

"1* 

%  Ss 

o  ^ 

o  « 

+>  0  X 

C  —  •    w 

C    cd 
3  .O 
.CJ     ^ 

if 

o 
1 

Calcareous   black 
grass-peat  
Sphagnum-covered 
peat    

6.40 
5.90 

2.940 
2.000 

42.81 
49.32 

0.600 
none 

28.71 
32.91 

14.56 
24.66 

Acid  ''muck"  soil    .  . 

5.60 

1.340 

14.58 

none 

7.14 

10.88 

Fargo   clay  loam.  .  .  . 
Fargo   silt  loam  

3.92 
14.89 

0.250 
0.823 

2.678 
10.02 

2.360 
0.200 

2.66 
9.91 

10.72 
12.17 

Carrington  silt  loam. 
Hempstead  silt  loam 
Prairie-covered   loess 
Forest-covered    loess 

6.22 
3.07 
7.89 
1.87 

0.371 
0.256 
0.301 
0.128 

4.733 
3.373 
3.704 
1.638 

0.090 
0.020 
0.240 
0.120 

4.95 
3.61 
3.40 
1.79 

12.76 
13.17 
12.30 
12.79 

1.  Analysis  of  "fibrin  from  blood"  hydrolyzed  in  the  presence 
of  100  grams  ignited  subsoil.  This  analysis  was  conducted  in  order 
to  ascertain  if  possible  the  effect  of  soil  minerals  upon  the  hydro- 
lysis of  a  pure  protein.  Fibrin  wras  selected  because  it  was  from 
a  sample  already  analyzed  (Gortner  1916  c).  The  subsoil  was  first 
ignited  to  redness  in  a  muffle  furnace  for  an  hour,  in  order  to  drive 
off  all  the  organic  matter,  and  subsequently  cooled  in  a  desiccator. 

Duplicate  portions  of  five  grams  fibrin  and  100  grams  ignited 
subsoil  were  hydrolyzed  in  the  presence  of  an  excess  of  hydrochlo- 
ric acid.  Upon  the  application  of  heat,  fumes  of  hydrogen  chloride 
were  evolved  for  some  time.  The  analysis  was  conducted  like 
that  of  the  mineral  soils,  excepting  that  a  600  cc.  aliquot  was  used 
for  the  analysis,  this  amount  of  solution  being  equivalent  to  three 
grams  of  fibrin.  After  making  the  ammonia  determination  the 
humin  precipitate  was  washed  by  decantation  in  the  usual  man- 
ner and  the  filtrate  made  to  250  cc.  volume.  The  first  wash  solu- 


39 

tion  from  the  humin  precipitate  had  a  characteristic  light  blue 
color  in  each  case.  Duplicate  Kjeldahl  determinations  were  made 
on  25  cc.  portions  of  this  solution  and  the  results  listed  as  total- 
nitrogen-in-the-filtrate-from-humin.  The  remaining  200  cc.  solu- 
tion was  used  for  the  precipitation  of  the  bases  and  the  subsequent 
analysis,  but  of  course  the  results  were  calculated  on  the  basis  of 
the  total  volume.  The  total  nitrogen  belonging  to  the  aliquot  an- 
alyzed was  determined  by  adding  the  nitrogen  obtained  as  am- 
monia, humin,  and  total-nitrogen-in-the-filtrate-from-humin  to  the 
insoluble-humin-nitrogen-in-the-soil.  The  filtrate-from-the-bases 
was  made  to  250  cc.  volume.  Twenty-five  grams  of  phosphotungstic 
acid  was  used  for  the  precipitation  of  the  bases.  The  nitrogen 
retained  by  the  barium  phosphotungstate  was  0.0022  gram  for 
Sample  I  and  0.0020  gram  for  Sample  II. 

The  experimental  data  showing  the  grams  of  nitrogen  found 
a-nd  per  cent  of  total  nitrogen  are  given  in  Table  II. 

Table  II. — Nitrogen  distribution  *in  tJyree  grams  of  Merck's  "fibrin 
from  blood"  hydrolyzed  in  the  presence  of  100  grams  of  ignited  subsoil. 


Grams  nitrogen          Per  cent  of  total  nitrogen 


I 

II 

1         I 

II        1 

Av. 

Total  \ 

04577 

04591 

Ammonia  N  •  •  

0.0457 

0.0455 

9.98 

9.91 

. 
9.95 

Insoluble  humin  N  in  soil 
Humin        N       pptd.       by 
CaCOH), 

0.0125 
00220 

0.0130 
0.0221 

2.73 
4.81 

2.83 
4.81 

2.78 
4.81 

Basic    N    •  •  
\rgmine  N  •  • 

0.1219 
0.0598 

0.0995 
0.0440 

26.63 
13.07 

21.68 
9.58 

24.15 
11.32 

Histidine^T 

none 

none 

none 

Lysine  N  •  •  

Cystine  N 

0.0597 

0.0531 


13.04 

11.57 

12.30 
0.51 

Amino  N  in  bases          •  • 


00775 

0.0746 

16.93 

16.25 

16.59 

N  in  filtrate  from  bases.. 
Amino    N    in   filtrate  from 
bases 

0.2725 
02671 

0.2909 
02702 

59.54 
58.36 

63.36 
58.85 

61.45 
58.61 

Non-amino    N    in    filtrate 
from  bases                •  • 

00054 

00207 

1  18 

451 

284 

Total    N    regained  ....-••• 

0.4746 

0.4710 

103.69 

102.59 

103.14 

iFrom  data  of  Gortner  (1916  c). 

Table  III  shows  a  comparison  of  these  analyses  with  other 
analysis  of  the  same  sample  of  fibrin  hydrolyzed  alone,  and  in  the 
presence  of  three  times  its  weight  of  cellulose  (data  of  Gortner 
1916  c). 

Differences  .between  these  analyses,  together  with  differences 
between  duplicates  in  each  series  of  analysis,  and  data  showing 
"maximum"  and  "average"  experimental  differences  to  be  expected 
are  given  in  Table  IV. 

These  comparisons  will  be  considered  in  detail  under  "Dis- 
cussion" in  the  latter  part  of  this  paper. 


40 


Table  III. — Comparative  analyses  of  three  grains*   of  Merck's 
'fibrin  from  blood"  hydrolyzed  alone  and  in  the  presence  of  carbo- 


hydrate and  of  ignited  subsoil. 


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I  Per  cent  total  jPer  cent  total]  Per  cent  total 
nitrogen    I         nitrogen     I          nitrogen 


Ammonia    N    

1015 

985 

995 

Humin  N  ..  .  . 

283 

7  72 

7  59 

Arginine  N                       •  . 

1091 

856 

11  32 

Histidine  N 

4  36 

4  86 

none 

Lysine  N  

1205 

11  04 

1230 

Cystine    N    .  . 

051 

071 

051 

Amino  N  in  filtrate  from  bases.  .  .  . 
Non-amino  N  in  filtrate  from  bases 
Total  N  regained  

55.43 
2.51 
98.75 

52.02 
3.91 
98.67 

58.61 
2.84 
103.14 

*The  three  gram  portion  contained  approximately  0.4550  gm.  nitrogen. 

Table  IV. — Difference  between  duplicate  analyses  (due  to  experi- 
mental errors),  the  differences  apparently  due  to  the  addition  of  carbo- 
hydrate and  of  ignited  subsoil,  as  well  as  Van  Slyke's  "maximum"  and 
<( average"  differences  to  be  expected. 


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Ammonia   N..  .  . 

0.01 

0.48 

0.07 

—0.30 

—0.20 

0.37 

0.12 

Humin   N  

0.11 

0.25 

0.10 

+4.89 

+4.76 

039 

020 

Arginine    N.  .  .  . 

0.73 

0.86 

3.49 

I      ••**J'' 

.    —2.35 

+0.41 

1.27 

0.73 

Histidine  N.  .  .  . 

0.07 

0.66 

none 

+0.50 

—4.36 

2.14 

0.79 

(0.93)1 

Lysine  N.     .... 

0.08 

0.11 

1.47 

1.01 

-f-0.25 

1.23 

0.61 

Cystine  N  

0.10 

0.00 

+0.20 

o'n 

Amino  N  in  fil- 

trate         from 

bases  

2.12 

2.15 

0.49 

—3.41 

+3.18 

1.60 

0.63 

Non-amino  N  in 

(0.60)1 

filtrate      from 

bases  ........ 

0.27 

0.40 

3.23 

+1.40 

+0.33 

1.20 

0.68 

2The   figure   in   parentheses   represents   the   second    greatest   difference   be- 
tween duplicates  observed  by  Van  Slyke   (1911). 


41 

2.  Calcareous  black  grass-peat.  Duplicate  samples  of  15 
grams  were  hydrolyzed  for  48  hours  in  the  presence  of  100  cc.  con- 
stant boiling  hydrochloric  acid,  and  the  analysis  conducted  as  de- 
scribed under  the  method  for  a  peat  soil. 

In  the  precipitation  of  the  phosphotungstic  acid  with  barium 
chloride,  it  was  difficult  to  determine  when  the  precipitation  was 
complete.  On  addition  of  barium  chloride  a  precipitate  formed  and 
the  pink  color  slowly  disappeared.  Both  sodium  hydroxide  and 
barium  chloride  were  repeatedly  added.  The  change  in  color  oc- 
curred several  times  during  a  half-hour.  At  all  times  test  portions 
gave  immediate  granular  precipitates  with  both  sodium  sulfate 
and  barium  chloride.  This  indicated  that  we  could  not  depend 
upon  the  test  with  sodium  sulfate  to  determine  when  all  the 
phosphotungstic  acid  was  precipitated.  After  the  addition  of  a 
large  amount  of  barium  chloride  the  solution  was  allowed  to  stand 
several  hours  in  order  to  allow  equilibrium  to  be  established.  At 
the  end  of  this  time  test  portions  gave  a  heavy  precipitate  with 
sodium  sulfate,  but  no  action  with  barium  chloride.  In  all  subse- 
quent work,  both  with  peat  and  mineral  soils,  the  addition  of  barium 
chloride  to  the  solution  of  the  phosphotungstates  was  extended 
over  an  hour  or  more  and  the  precipitate  then  allowecl  to  settle 
over  night. 

This  barium  phosphotungstate  precipitate  was  washed  thor- 
oughly by  decantation  with  hot  water  until  free  of  chlorides.  This 
operation  was  carried  out  in  the  original  beaker,  and  in  the  case  of 
many  mineral  soils  required  from  500  to  1000  cc.  to  remove  all 
chlorides.  The  washed  precipitate  of  barium  phosphotungstate  and 
filter  on  kjeldahling  gave  0.0021  gram  nitrogen  retained  by  Sample 
I  and  0.0055  gram  by  Sample  II. 

The  experimental  data  showing  the  grams  of  nitrogen  and 
per  cent  of  tgtal  nitrogen  are  given  in  Table  V. 

Table  V . — Nitrogen  distribution  in  calcareous  black  grass-peat. 


Grams  nitrogen     |  Per  cent  of  total  nitrogen 


I 

II 

I 

II       1 

Av. 

Total  N 

04412 

04412 

Ammonia  N 

00833 

00867 

1888 

1965 

1926 

Humin   N    

0.1145 

0.1155 

25.95 

26.18 

26.07 

Basic  N 

00494 

0  0441 

11  20 

1000 

1060 

Basic   N   set   free   as   NH3  by 
50%   KOH    ..  .  

0.0150 

0.0139 

3.40 

3.15 

3.27 

Basic  N  not  set  free  as  NHa 
by  50%   KOH......  
Amino  N  of  bases  

0.0344 
0.0309 

0.0302 
0.0251 

7.80 
7.00 

6.85 
5.69 

7.33 
6.35 

Non-amino  N  of  bases  •  .  .    . 

0.0185 

0.0190 

4.19 

4.31 

4.25 

N  in  filtrate  from  bases  
Amino     N     in     filtrate     from 
bases  
Non-amino  N  in  filtrate  from 
bases                .... 

0.1932 
0.1810 
0  0122 

0.1810 
0.1672 
00138 

43.79 
41.02 
277 

41.02 
37.90 
3  12 

42.40 
39.46 
2.94 

Total  N  regained  

0.4404 

0.4273 

99.82 

96.85 

98.33 

42 

3.  Sphagnum-covered  peat.  Duplicate  15  gram  samples  were 
hydrolyzed  in  the  usual  manner.  It  was  found  in  the  ammonia  de- 
termination that  all  of  the  ammonia  nitrogen  could  not  be  driven 
off  in  a  half-hour  when  the  volume  of  solution  was  large  and  a 
bulky  precipitate  of 'iron  and  aluminum  hydroxides  was  present. 
Continued  distillation  for  a  further  half-hour  in  this  case  gave 
additional  ammonia  nitrogen  amounting  to  0  0060  gram.  In  all  sub- 
sequent work  with  both  peats  and  soils  the  volume  was  kept  smaller 
by  the  use  of  a  more  concentrated  suspension  of  calcium  hydroxide. 

The  calcium  hydroxide  precipitate  containing  the  "humin" 
nitrogen  was  difficult  to  digest  in  the  subsequent  Kjeldahl  deter- 
mination, due  to  the  large  amount  of  carbonaceous  organic  material 
present.  From  100-200  cc.  of  sulfuric  acid  were  required  for 
the  digestion.  After  digestion  the  material  was  transferred  to  a 
1000  cc.  flask  and  250  cc.  portions  used  for  the  distillation.  It  was 
often  necessary  to  heat  the  flask  on  a  sand  bath  to  prevent  severe 
bumping.  This  trouble  was  obviated  in  later  work  by  adding  zinc 
dust  instead  of  granulated  zinc  to  the  distilling  flask.  The  nitrogen 
retained  by  the  barium  phosphotungstate  amounted  to  0.0009  gram 
in  Sample  I,  and  0.0013  gram  in  Sample  II. 

The  experimental  data  showing  the  grams  of  nitrogen  and 
per  cent  of  total  nitrogen  are  given  in  Table  VI. 

Table  VI. — Nitrogen  distribution  in  sphagnum-covered  peat. 

Grams  nitrogen      |    Per  cent  of  total  nitrogen 


1         I 

1         II 

I         1 

II 

Av. 

Total  N 

03000 

03000 

Ammonia  N 

00659 

00737 

21  97 

2457 

2327 

Humin   N    

0.0779 

0.0804 

25.96 

26.80 

26.38 

Basic  N  

0.0281 

0.0303 

9.37 

10.10 

9.73 

Basic    N   set   free   as    NH3 
by  50%  KOH.......... 
Basic    N    not    set   free    as 

NH3  by  50%  KOH.  .    .  . 

0.0091 
00190 

0.0088 
00?15 

3.03 
632 

2.93 

7  17 

2.98 
675 

Amino  N  of  bases  
Non-amino  N  of  bases... 
N  in  nitrate  from  bases.. 
Amino   N   in   filtrate   from 
bases  . 

0.0171 
0.0110 
0.1396 

0  1292 

0.0145 
0.0158 
0.1184 

01135 

5.70 
3.67 
46.53 

43.06 

4.83 
5.27 
39.46 

37.83 

5.26 
4.47 
43.00 

40.45 

Non-amino    N    in    filtrate 
from  bases   

0.0104 

0.0049 

3.47 

1.63 

2.55 

Total  N  regained  

0.3115 

0.3028 

103.83 

100.93 

102.38 

4.  Acid  "muck"  soil  One  25  gram  sample  was  hydrolyzed 
in  the  presence  of  125  cc.  concentrated  hydrochloric  acid  for  48 
hours.  The  -ammonia  nitrogen  was  determined  as  usual,  and  the 
precipitate  of  calcium  hydroxide  containing  the  '"humin"  was 
washed  in  a  taM  soil  beaker  in  the  manner  described  under  the 
method  for  a  mineral  soil.  After  digestion  of  this  precipitate,  the 
material  in  the  !£jeldahl  flask  was  diluted  to  500  cc.  and  duplicate 
distillations  made  on  250  cc.  portions.  The  bases  were  precipitated 
with  15  grams  phosphotungstic  acid.  The  total  nitrogen  retained  by 
the  barium  phosphotungstate  was  0.0022  gram.  The  solution  of 
the  filtrate-from-the-bases  was  made  to  a  volume  of  250  cc. 


43 


The  experimental  data  showing  the  grams  of  nitrogen  and  per 


cent  of  total  nitrogen  are  found  in  Table  VII. 


Table  VII. — Nitrogen  distribu  tion  in  an  acid  "muck"  soil. 


Grams 
nitrogen 

Per  cent 
of  total 
nitrogen 

Total  N 

Ammonia  N  

Humin    N 

Basic  N   

Basic  N  set  free  as  NH3  by  50%  KOH 

Basic  N  not  set  free  as  NH3  by  50%  KOH 

Ammo  N  of  bases 

Non-amino   N  of  bases 

N  in  filtrate  from  bases 

Ammo  N  in  filtrate  from  bases 

Non-amino  N  in  filtrate  from  bases • 

Total  N  regained 


0.3350 
0.0653 
0.0925 
0.0454 
0.0104 
0.0350 
0.0306 
0.0148 
0.1300 
0.1133 
0.0167 
0.3332 


19.49 
27.61 
13.55 

3.10 
10.45 

9.13 

4.42 
38.81 
33.82 

4.99 
99.46 


5.  Fargo  clay  loam.  Duplicate  portions  of  250  grams  were 
hydroh  zed  for  48  hours.  The  "hitinin"  precipitate  required  100  cc. 
sulfuric  acid  for  the  digestion.  After  digestion  the  material  was 
transferred  to  a  500  cc.  flask  and  21>0  cc.  portions  used  for  the  dis- 
tillation. This  method  was  followed  subsequently  with  th'e  "humin" 
nitrogen  determination  of  all  the  mineral  soils. 

The  nitrate  from  "humin"  was  of  a  sirupy  consistency  in  each 
case.  The  first  addition  of  15  grams  of  phosphotungstic  acid  did 
not  entirely  precipitate  the  bases.  Five  gram  portions  were  added 
from  time  to  time  until  a  total  of  50  grams  had  been  used.  After 
standing  the  usual  length  of  time  the  precipitate  of  the  bases  was 
filtered  off,  but  even  then  the  wash  water  caused  the  formation  of 
a  small  additional  precipitate  in  the  filtrate.  After  warming  on  the 
steam  bath  this  final  solution  was  perfectly  clear,  and  on  standing 
over  night,  only  a  trace  of  precipitate  was  formed  so  the  precipita- 
tion was  considered  complete.  It  appears  probable  that  a  portion 
of  this  precipitate  is  due  to  the  formation  of  inorganic  phospho- 
tungstates  which  consume  a  very  large  amount  of  the  phospho- 
tungstic acid,  for  if  all  of  this  precipitate  had  consisted  o.f  basic 
nitrogen  compounds  the  amount  of  nitrogen  recovered  should  have 
been  greater  than  the  amount  which  was  actually  found.  In  all 
the  subsequent  work  35  grams  of  phosphotungstic  acid  was  used 
for  the  precipitation  of  the  bases  in  the  hydrolysates  from  mineral 
soils. 

The  phosphotungstate  precipitate  dissolved  very  slowly  in  the 
sodium  hydroxide  as  did  all  other  phosphotungstic  acid  precipitates 
of  the  mineral  soils  studied.  A  Kjeldahl  determination  of  the 
barium  phosphotungstate  gave  0.0026  gram  nitrogen.  The  solution 
containing  the  nitrogen  of  the  bases  was  made  up  to  100  cc.  volume. 

During  the  concentration  of  the  filtrate  from  the  bases  so  much 
precipitate  separated,  that  this  was  filtered  off  and  the  solution 
made  up  to  300  cc.  volume.  The  salt  remaining  was  dissolved  in 
water  and  also  made  up  to  a  volume  of  300  cc.  Aliquot  portions 
were  taken  from  each  solution  and  combined  for  the  different  de- 
terminations. 


The  experimental  data  giving  the  grams  of  nitrogen  and  per 
cent  of  total  nitrogen  are  found  in  Table  VIII. 

Table  VIII. — Nitrogen  distribution  in  Fargo  clay  loam. 


Grams  nitrogen      [    Per  cent  of  total  nitrogen 


II 


II  Av. 


Total  N 

03368 

0  3338 

Ammonia    N 

0  0788 

"00821 

23  40 

24  60 

24  (X) 

Insoluble  humin  N  in  soil. 
Humin   N  pptd.  by 
Ca(OH)2           

0.0948 
00352 

0.0948 
00266 

28.15 
1045 

28.40 
797 

28.27 
Q  21 

Basic    N    . 

i 

00320 

9  58 

Basic    N   set   free   as    NH3 
by  50%  KOH  

0.0108 

3?3 

323 

Basic    N    not    set    free    as 
NH3  by  50%  KOH 

00212 

635 

63=1 

Amino  N  of  bases          •  • 

00215 

6  44 

Non-amino  N  of  bases     •  • 

00105 

3  14 

N  in  filtrate  from  bases.  .  . 

0.1092 

32.71 

Amino   N   in   filtrate   from 
bases 

0  1027 

3077 

30  77 

Non-amino    N    in    filtrate 

00065 

1  95 

1  9S 

Total  N  regained  

0.3447 

103.26 

103.77 

1  Entire   sample   lost  during  precipitation   of  bases. 

6.  Fargo  silt  loam.  Two  125  gram  portions  were  hydrolyzed 
for  48  hours  with  500  cc.  hydrochloric  acid  (sp.  gr.  1.18).  During 
the  first  few  hours  large  amounts  of  hydrogen  chloride  were 
evolved. 

The  resulting  hydrolysates  from  the  two  flasks  were  com- 
bined and  diluted  to  2  liters.  After  settling,  a  1  liter  portion  was 
syphoned  off  and  analyzed  by  the  usual  method.  Two  100  cc.  por- 
tions were  used  for  the  determination  of  total  nitrogen  in  solu- 
tion, and  another  portion  of  200  cc.  was  used  for  determination 
of  the  "Jodidi  numbers."  The  nitrogen  retained  by  the  barium 
phosphotungstate  was  0.0091  gram.  The  solution  containing  the 
basic  nitrogen  was  made  to  100  cc.  volume  and  that  containing 
the  nitrogen  in  the  filtrate  from  the  bases  to  300  cc.  volume. 

The  experimental  data  showing  grams  of  nitrogen  and  per 
cent  of  total  nitrogen  are  given  in  Table  IX. 

Table  IX. — Nitrogen  distribution  in  Fargo  silt  loam. 


Total  N 

Ammonia  N 

Insoluble  humin  N  in  soil 

Humin  N  pptd.  by  Ca(OH)2 

Basic  N 

Basic  N  set  free  as  NH3  by  50%  KOH 

Basic  N  not  set  free  as  NH3  by  50%  KOH 

Amino  N  of  bases •  • •  • 

Non-amino  N  of  bases •  • 

N  in  filtrate  from  bases • 

Amino  N  in  filtrate  from  bases 

Non-amino  N  in  filtrate  from  bases 

Total  N  regained   


Grams 
nitrogen 


Per  cent 
of  total 
nitrogen 


0.9238 
0.2454 
0.2118 
0.03011 
0.1119 
0.0296 
0.0823 
0.0695 
0.0424 
0.3246 
0.3015 
0.0231 


26.56 

22.93 

3.26 

12.11 

3.20 

8.91 

7.52 

4.59 

35.14 

32.64 

2.50 

100.002 


By  difference. 


Calculated. 


45 

7.  Carrington  silt  loam.  Sample  I  (Nerstrand  origin).  The 
hydrolysis  of  250  grams  was  carried  out  according  to  the  method 
described  under  Fargo  silt  loam.  The  "humin"  nitrogen  was  de- 
termined as  outlined  under  Fargo  clay  loam.  Sample  II  (Morris- 
town  origin)  was  hydrolyzed  and  the  same  methods  of  analysis 
employed  throughout  as  was  applied  to-  Sample  I.  The  nitrogen 
retained  by  the  barium  phosphotungstate  precipitate  was  0.0028 
gram  in  Sample  I  and  0.0025  gram  in  Sample  II.  In  both  samples 
the  solutions  containing  the  bases  and  nitrate  from  the  bases  were 
made  to  the  same  dilution  as  under  Fargo  silt  loam. 

The  experimental  data  showing  grams  of  nitrogen  and  per  cent 
of  total  nitrogen  are  given  in  Table  X. 

Table  X. — Nitrogen  distribution  in  Carrington  silt  loam. 

I        Sample  No.  I       |     Sample  No.  II 


Grams 
nitrogen 

Per  cent 
of  total 
nitrogen 

Grams 
nitrogen 

Per  cent 
of  total 
nitrogen 

Total  N               .... 

0  5124 

04920 

Ammonia    N    

0  1463 

28  55 

01381 

2807 

Insoluble  humin  N  in  soil  
Humin  N  pptd.  by  Ca(OH)2.  •  
Basic    N    

0.1324 
0.0304 
00600 

25.84 
5.93 
11  71 

0.1210 
0.03263 
00725 

24.59 
6.63 
1474 

Music    N    set    free    as    NPL    by    50% 
KOH    

0.0172 

3  36 

00156 

3  17 

Masic  N  not  set  free  as  NH;t  by  50% 
KOH    ..        ... 

004?8 

835 

00569 

11  S7 

Amino  N  of  bases  

0.0412 

804 

0  0393 

799 

Non-amino  N  of  bases  

00188 

367 

0033? 

67S 

N  in  filtrate  from  liases  

0.1305 

25.47 

0  1390 

28.25 

Amino  N  in  filtrate  from  bases  
Non-amino  N  in  filtrate  from  bases. 
Total    N   regained  •  

i 
0.4996 

97.50 

0.1272 
0.0118 
0.5032 

25.85 
2.40 
102.28 

Solution  lost   at   this  point. 
2  Result    calculated    from    the    digestion    of   one-half    the    precipitate. 

8.  Hempstead  silt  loam.  Duplicate  250  gram  samples  were 
hydrolyzed  for  48  hours.  The  "humin"  was  washed  and  the  de- 
termination carried  out  as  described  under  mineral  soils.  The  only 
explanation  which  occurs  for  the  higher  ammonia  nitrogen  of  one 
hydrolysate  is  that  Sample  I  must  have  been  heated  more  strongly 
during  hydrolysis.  From  unpublished  experiments  conducted  at 
this  Station  it  has  been  found  that  when  a  pure  protein  is  hydro- 
lyzed under  vigorous  boiling,  a.  greater  amount  of  ammonia  nitro- 
g~en  is  invariably  obtained  than  when  hydrolyzed  under  slow  boil- 
ing conditions.  This  observation  was  made  subsequently  to  my 
own,  so  that  the  importance  of  the  rate  of  boiling  was  not  known 
at  the  time  of  doing  my  work. 

Only  25  grams  of  phosphotungstic  acid  were  added  for  the 
precipitation  of  the  bases.  After  standing  the  solution  gave  a 
further  precipitate  on  addition  of  more  phosphotungstic  acid.  It 
was  concluded,  however,  that  the  organic  bases  were  entirely  pre- 
cipitated, for  on  washing  the  precipitate  with  the  phosphotungstic 
acid  wash  water  no  additional  precipitate  formed.  The  nitrogen 
determination  gave  0.0011  gram  retained  by  the  barium  phospho- 
tungstate. The  solution  containing  the  nitrate  from  the  bases  was 
made  to  300  cc.  volume. 


46 

The  experimental  data  giving  the  grams  of  nitrogen  and  pel 
cent  of  total  nitrogen  will  be  found  in  Table  XI. 

Table  XL — Nitrogen  distribution  in  Hemp  stead  silt  loam. 

|      Grams  nitrogen      |    Per  cent  of  total  nitrogen 
|         I~  II   "  I  II        |      Av. 


Total   "NT 

0  3416 

03485 

01042 

0.0989 

30.50 

28.38 

29.44 

Insoluble  humin   N  in  soil 
Humin  N  pptd.  by 
Ca(OH)2     

0.0981 
0.0164 

0.0920 
i 

28.72 
4.80 

26.40 

27.56 
4.80 

Basic  N 

00389 

11  39 

Basic   N   set  free   as   NH3 
by  50%  KOH 

00078 

2.28 

2.28 

Basic    N    not    set    free    as 
NH3  by  50%  KOH 

00311 

9  10 

9.10 

00254 

744 

00135 

395 

N  in  filtrate  from  bases 

00960 

28.10 

Amino   N  in   filtrate   from 
bases         

0.0839 

24.56 

24.56 

Non-amino    N    in    filtrate 
from  bases 

00121 

3.54 

3.54 

Total  N  regained  ~.  

0.3536 



103.51 

101.29 

1  Solution   lost  at  this   point. 

9.  Prairie-covered  loess.  In  Sample  I,  250  grams  were  hy- 
drolyzed  in  the  presence  of  250  cc.  of  constant  boiling  hydrochloric 
acid  for  48  hours.  The  hydrolysate  on  cooling  was  diluted  to  1000 
cc.  and  a  500  cc.  portion  was  syphoned  off  and  used  for  the  an- 
alysis. In  Sample  II,  two  125  gram  samples  were  hydrolyzed  in 
the  same  manner  as  outlined  under  Fargo  silt  loam  (500  cc.  con- 
stant boiling  hydrochloric  acid  to  125  grams  soil).  The  dilution 
and  aliquot  used  for  analysis  were  also  the  same.  The  nitrogen 
retained  by  the  barium  phosphotungstate  amounted  to  0.0020  gram 
in  Sample  I,  and  0.0072  gram  in  Sample  II.  The  solution  con- 
taining the  bases  was  diluted  to  100  cc.  in  both  samples. 

The  experimental  data  showing  grams  of  nitrogen  found  and 
per  cent  of  total  nitrogen  are  given  in  Table  XII. 

Table  XII. — Nitrogen  distribution  in  prairie-covered  loess. 

|  Grams  nitrogen  |  Per  cent  of  total  nitrogen 


! 

I 

II 

i 

II 

Av. 

Total  N 

03907 

04012 

Ammonia  N  •  • 

0  1223 

0  1194 

31  30 

29.76 

30.53 

Insoluble  humin  N  in  soil 
Humin    N    pptd.   by 
Ca(OH)2 

0.0922 
00198 

0.1007 
0.0213 

23.60 
5.07 

25.10 
5.31 

24.35 
5.19 

Basic  N  

0.0444 

0.0562 

11.36 

14.01 

12.68 

Basic    N   set   free   as    NH3 
by  50%  KOH   
Basic    N    not    set    free    as 
NH3  by  50%  KOH  
Amino  N  of  bases   

0.0100 

0.0344 
0.0271 

0.0132 

0.0430 
0.0322 

2.56 

8.80 
6.93 

3.29 

10.72 
8.03 

2.92 

9.76 
7.48 

Non-amino  N  of  bases... 
N  in  filtrate  from  bases.. 
Amino   N   in   filtrate  from 
bases 

0.0173 
0.1152 

0  1044 

0.0240 
0.1128 

0  1033 

4.43 
29.49 

26.72 

5.98 
28.12 

25.75 

5.20 
28.80 

26.24 

Non-amino    N    in    filtrate 
from  bases   

0.0108 

0.0095 

2.76 

2.37 

2.56 

Total  N  regained   

0.3939 

0.4104 

100.82 

102.30 

101.56 

47 

10.  Forest-covered  loess.  In  Sample  1,  300  grams  of  soil 
were  hydrolyzed  and  diluted  in  the  same  manner  as  Sample  I  of 
prairie-covered  loess.  In  Sample  II,  300  grams  of  soil  were  hy- 
drolyzed under  the  same  conditions  as  Sample  II  of  prairie-covered 
loess.  The  nitrogen  retained  by  the  barium  phosphotungstate  was 
0.0024  gram  in  Sample  I,  and  0.0023  gram  in  Sample  II.  The 
solution  containing  the  nitrogen  of  the  bases  was  Diluted  to  100 
cc.  in  both  samples  and  the  solutions  containing  the  total  nitrogen 
of  the  filtrates  were  made  to  a  volume  of  300  cc. 

It  is  observed  that  the  volume  of  acid  used  in  the  hydrolysis 
had  little  effect  on  the  proportion  of  the  different  fractions.  The 
only  observed  difference  is  in  the  insoluble  humin  nitrogen  retained 
by  the  soil  residue,  and  this  is  slightly  larger  in  Sample  II,  which 
was  hydrolyzed  in  the  presence  of  the  greatest  excess  of  acid.  In 
connection  with  this  it  must  also  be  noted  that  there  is  a  somewhat 
larger  quantity  of  nitrogen  in  solution  in  Sample  II  than  in  Sample 
I.  Much  the  same  results  are  shown  with  the  prairie-covered  loess. 
All  increases  or  decreases  in  the  various  fractions  due  to  the  greater 
excess  of  acid  may  well  be  considered  to  be  within  the  experimental 
error. 

The  experimental  data  showing  grams  of  nitrogen  found  and 
per  cent  of  total  nitrogen  are  given  in  Table  XIII. 

Table  XIII. — Nitrogen  distribution  in  forest-covered  loess. 


|       Grams  nitrogen      |    Per  cent  of  total  nitrogen 

]         I 

II 

I 

II        [     Av. 

Total  N  

0.2224 
0.0646 
0.0574 

0.0140 
0.0316 

0.00/4 

0.0242 
0.0175 
0.0141 
0.0621 

0.0585 

0.0036 
0.2297 

0.2362 
0.0669 
0.0662 

0.00801 
0.0325 

0.0096 

0.0229 
0.0172 
0.0153 
0.0626 

0.0568 
0.0058 

Ammonia  N   

29.05 
25.81 

6.29 
14.21 

3.33 

10.88 
7.87 
6.34 
27.92 

26.30 

1.62 
103.28 

28.32 
28.03 

3.30 
13.76 

4.06 

9.70 

7.28 
6.48 
26.50 

24.05 
2.45 

U/O.UO2 

28.69 
26.92 

4.84 
13.98 

3.69 

10.29 
7.57 
,  6.41 
27.21 

25.17 

2.04 
101.64 

Insoluble  humin  N  in  soil 
Humin   N  pptd.  by 
Ca(OH)2     

Basic  N  

Basic   N   set   free  as   NH3 
by  50%  KOH  

Basic    N    not    set    free    as 
NH3  by  50%  KOH 

Amino  N  of  bases  

Non-amino  N  of  bases.  .  .  . 
N  in  filtrate  from  bases.. 
Amino   N   in   filtrate   from 
bases 

Non-amino    N    in    filtrate 
from  bases 

Total   N   regained  

By    difference 


Calculated. 


1 1 .  Sphagnum-covered  peat  hydrolyzed  in  the  presence  of  nine 
times  its  weight  of  a  mineral  subsoil.  Duplicate  10  gram  samples 
were  hydrolyzed  in  the  presence  of  90  grams  subsoil  with  constant 
boiling  hydrochloric  acid  for  48  hours.  The  hydrolysate  was  con- 
centrated as  much  as  possible  and  ammonia  nitrogen  determined 
on  the  entire  mixture  by  distillation  with  an  excess  of  calcium  hy- 
droxide for  one  hour.* 

*This  was  the  first  attempt  to  determine  the  nitrogen  fractions  in  the 
presence  of  a  mineral  soil.  For  certain  reasons  later  analyses  have  already 
been  reported  in  this  paper.  The  analyses  as  reported  in  this  paper  are 
by  no  means  in  chronological  order,  which  may  explain  seeming1  inconsist- 
encies. 


48 

The  residue  remaining  after  hydrolysis  was  washed  free  of 
chlorides  on  ordinary  funnels.  It  was  then  digested  in  three 
Kjeldahl  flasks  using  a  total  of  30  grams  potassium  sulfate  and 
360  cc.  sulfuric  acid.  After  digestion  the  material  was  transferred  to 
a  1000  cc.  flask,  made  to  volume,  and  the  nitrogen  content  deter- 
mined as  described  under  sphagnum-covered  peat.  It  will  be  ob- 
served that  the  total  "humin"  nitrogen  is  determined  here  instead 
of  being  reported  in  two  separate  fractions  as  was  done  in  the 
case  of  all  other  analyses  containing  mineral  soil. 

The  combined  filtrate  and  washings  from  the  "humin"  precipi- 
tate were  concentrated  in  the  usual  manner  and  made  to  200  cc. 
volume.  Duplicate  Kjeldahl  determinations  were  made  on  25  cc. 
portions  of  this  solution  and  the  results  listed,  as  total  nitrogen- 
in-the-filtrate-from-humin.  The  remaining  150  cc.  portion  was 
used  for  the  precipitation  of  the  bases,  the  subsequent  procedure 
being  completed  as  described  in  the  "fibrin  from  blood"  analysis. 
The  barium  phosphotungstate  precipitate  retained  0.0016  gram  of 
nitrogen  in  Sample  I,  and  0.0053  gram  in  Sample  II. 

In  the  second  sample  the  combined  filtrate  and  washings 
from  the  phosphotungstic  acid  precipitate  of  the  bases  were  brought 
to  near  the  neutral  point  with  50  per  cent  sodium  hydroxide  and  a 
small  amount  of  acetic  acid  added  at  once.  After  the  addition  of 
the  acid  it  seemed  possible  that  the  neutral  point  had  not  been 
reached  the  first  time,  so  more  sodium  hydroxide  was  added  until 
the  neutral  point  was  just  passed  and  acetic  acid  again  added.  The 
resulting  solution  was  placed  in  a  double-necked  distilling  flask 
and  an  attempt  made  to  concentrate  the  solution  under  diminished 
pressure.  Frothing  was  so  intense  that  it  was  impossible  to  effect 
any  concentration.  When  alcohol  was  added  the  distillation  con- 
tinued quietly  as  long  as  any  alcohol  was  present,  but  after  that  the 
frothing  continued.  The  mixture  behaved  like  a  concentrated  soap 
solution.  The  solution  was  finally  concentrated  in  an  evaporating 
dish  over  a  water-bath  and  the  resulting  solution  made  to  300  cc. 
volume.  On  shaking  the  solution  frothed  very  badly. 

The  experimental  data  showing  the  grams  of  nitrogen  found 
and  the  per  cent  of  total  nitrogen  are  given  in  Table  XIV. 

A  comparison  between  these  analyses  and  those  of  the  peat 
hydrolyzed  alone  is  made  in  Table  XV,  the  data  of  the  peat  hy- 
drolyzed  alone  being  taken  from  Table  VI,  and  recalculated  from 
a  15  gram  basis  to  a  10  gram  basis. 


49 


Table    XI]7. — Nitrogen    distribution    in    sphagnum-covered   peat 
livdrolyscd  in  the  presence  of  nine  times  its  weight  of  a  mineral  subsoil. 


Grams  nitrogen 
I  II" 


I    Per  cent  of  total  nitrogen 
"  I          |       "II        |     Av. 


Total   N 

02441 

02441 

Ammon'a  N  

0.0625 

0.0621 

25.60 

25.44 

25.52 

Humin    N            

0.0658 

0.07561 

26.96 

j 

26.96 

Basic  N   •  • 

00268 

0  0374' 

1098 

] 

10.98 

Basic    N   set   free   as    NH3 
by  50%  KOH    
Basic    N    not    set    free    as 
NH3  by  50%  KOH 

0.0095 
00173 

0.0105 

3.89 
709 

4.30 

4.09 

\mino  N  of  bases  •  • 

00177 

00179 

725 

733 

7.29 

00091 

373 

N  in  filtrate  from  bases.. 
Amino   N   in   nitrate    from 
bases    •  • 

0.0954 
0.0836 

0.0948 
0.0815 

39.08 
34.25 

38.84 
33.39 

38.96 
33.82 

Non-amino     N     in    filtrate 
from    bases     
Total  N  regained   

0.0118 
0.2505 

0.0133 
^_..._: 

4.83 
102.62 

5.45 

5.14 
102.42 

1  These   results   are   evidently   incorrect   and    in    the    "average"    column   the 
figures  from   the  first  column  only   are  used. 

Table  XV. — Comparative  analyses  of  sphagnum- cohered  peat 
hydrolyzed  alone  and  in  the  presence  of  nine  times  its  zveight  of  a  min- 
eral subsoil. 


Grams  Nitrogen 

Apparent 
distribution 

of  Nin 
subsoil    in 
per  cent  of 
total 
nitrogen 

Peat 

Peat 
Subsoil1 

fncrease  (  +  ) 
or 
Decrease(—  ) 

Total  N 

0.2000 
0.0466 
0.0527 
0.0195 

0.0060 

0.0135 
0.0105 
0.0090 
0.0860 

0.0776 

0.0084 
0.2048 

0.2441 
0.0623 
0.0658 
0.0268 

0.0100 

0.0168 
0.0178 
0.0090 
0.0951 

0.0825 

0.0126 
0.2500 

+0.0441 
-J-0.0157 
+0.0131 
+0.0073 

+0.0040 

+0.0033 
+0.0073 

\mrr>.onia  N   .  .        

35.60 
29.71 
16.55 

9.07 

7.48 
16.55 

Humin   N 

Basic  N 

Basic  N  set  free  as  NH3  by 
50%   KOH 

Basic  N  not  set  free  as  NH3 
by  50%  KOH  
Amino  N  of  bases  
\on-amino  N  of  bases. 
N  in  filtrate  from  bases.... 
AmJ.no    N    in    filtrate    from 
bases    •  • 

+0.0091 
+0.0049 

+0.0042 
+0.0452 

20.64 
11.11 

9.52 
102.50 

Non-amino  N  in  filtrate  from 
bases  

Total  N  regained  .......... 

1  Ninety    gm.    of    subsoil    contained    0.0441    gm.    of    soil    nitrogen. 

12.  Sphagnum-covered  peat  hydrolyzed  in  the  presence  of 
metallic  tin.  This  peat  was  hydrolyzed  in  the  presence  of  a  reduc- 
ing agent  because  it  was  thought  that  the  amount  of  "humin"  nit- 
rogen would  be  reduced,  for  according  to  Samuely  (1902)  the 
formation  of  this  dark  colored  product  is  due  to  an  oxidative  proc- 
ess. Hlasiwetz  and  Habermann  (1871  and  1873)  hydrolyzed  pro- 
tein with  hydrochloric  acid  in  the  presence  of  stannous  chloride  in 
order  that  the  solution  should  remain  colorless.  Cohn  (1896-97 


50 

and  1898-99)  believed  that  the  use  of  »a  reducing  agent  was  not 
essential,  but  according  to  Otori  (1904)  this  is  a  mistake. 

It  is  perhaps  significant  that  the  "humin"  nitrogen  was  reduced 
to  3.90  per  cent  by  the  presence  of  a  reducing  solution.  It  is  not 
known  whether  there  was  sufficient  tin  present  to  maintain  a  reduc- 
ing solution  throughout  the  hydrolysis  inasmuch  as  the  ferric  iron 
in  the  peat  would  have  an  oxidizing  action  on  the  stannous  salt. 
The  sample  was  known  to  contain  iron  but  the  amount  was  not 
determined. 

Duplicate  15  gram  samples  were  hydrolyzed  with  100  cc.  hydro- 
chloric acid  (sp.  gr.  1.115)  for  48  hours  in  the  presence  of  five  and 
ten  grams  of  tin  respectively.  The  tin  was  first  partially  dissolved 
in  the  acid  before  the  samples  of  peat  were  added.  The  deter- 
mination of  "humin"  nitrogen  was  carried  out  as  directed  under 
sphagnum-covered  peat  excepting  that  the  digested  material  was 
diluted  to  500  cc.  instead  of  to  1  liter.  The  nitrogen  retained  by 
the  barium  phosphotungstate  was  0.0023  gram  in  Sample  I;  and 
0.0047  gram  in  Sample  II.  The  solution  containing  the  filtrate  from 
the  bases  was  diluted  to  a  volume  of  300  cc.  The  experimental  data 
giving  the  grams  of  nitrogen  found  and  the  per  cent  of  total 
nitrogen  are  given  in  Table  XVI. 


Table    XV L — Nitrogen    distribution    in 
hydrolyzed  in  the  presence  of  metallic  tin. 


sphagnum- covered    peat 


Grams  nitrogen      |    Per  cent  of  total  nitrogen 

1         I    . 
5  gm.  tin 

II 

10  gm.  tin 

I 

II 

Av. 

Total  N 

0.3000 
0.0633 
0.0699 
0.0320 

0.0099 

0.0221 
0.0193 
0.0127 
0.1  4041 

0.1297 

0.0107 
0.3056 

0.3000 
0.0578 
0.0650 
0.0417 

0.0100 

0.0317 
0.0219 
0.0198 
0.1380 

0.1307 

0.0073 
0.3025 

Ammonia  N  
Humin   N    

21.10 
23.30 
10.67 

3.30 

7.37 
6.43 
4.23 
46.80 

43.23 

3.56 
101.86 

19.27 
21.67 
13.90 

3.33 

10.57 
7.30 
6.60 
46.00 

43.57 

2.43 
100.83 

20.18 
22.48 
12.28 

3.31 

8.97 
6.86 
5.41 
46.40 

43.40 

2.99 
101.34 

Basic  N                    .... 

Basic   N   set  free  as   NH3 
by  50%  KOH   
Basic    N    not    set    free    as 
NH3  by  50%   KOH.... 
Amino  N  of  bases  '   •  • 

Non-amino  N  of  bases.  .  .  . 
N  in  filtrate  from  bases.. 
Amino   N   in  filtrate   from 
bases     

Non-amino    N    in    filtrate 
from  bases 

Total  N  regained  

1  This    result    is    from    a    single    determination    of    nitrogen. 

13.  Analysis  of  a  1  per  cent  hydrochloric  acid  extract  of  sphag- 
num-covered peat  and  (in  part)  of  calcareous  black  grass-peat. 
Acid  extraction  was  made  of  the  two  peats  in  direct  contact  with 
1  per  cent  hydrochloric  acid.  For  the  extraction  125  gram  por- 
tions were  placed  in  2.5  liter  acid  bottles  and  two  liters  of  1  per 
cent  acid  added.  In  the  case  of  calcareous  black  grass-peat,  how- 
ever, the  calculated  amount  of  hydrochloric  acid  necessary  to  neu- 
tralize the  calcium  oxide  was  first  added  and  then  sufficient  dilute 
acid  to  make  two  liters  of  a  1  per  cent  solution.  Five  hundred 


51 

grams  were  taken  in  the  case  of  sphagnum-covered  peat,  while  750 
grams  were  taken  in  the  case  of  calcareous  black  grass-peat.  The 
bottles  were  shaken  at  intervals  during  five  days  and  then  the  con- 
tents filtered  through  two  thicknesses  of  a  good  grade  of  cheese 
cloth  and  squeezed  in  the  hands.  The  resulting  solution  was  then  j 
filtered  through  two  thicknesses  of  filter  paper  on  a  Buchner  funnel. 

This  extract  was  colored  in  each  case  but  the  calcareous  black 
grass-peat  gave  a  deeper  straw  colored  solution  than  did  the  sphag- 
num-covered peat.  This  was  probably  due  to  the  presence  of  a 
larger  amount  of  iron  in  the  one  case  than  in  the  other.  The  cal- 
careous black  grass-peat  is  known  to  contain  a  very  considerable 
quantity  of  iron.  The  wash  water  in  both  instances  was  also  straw 
colored. 

It  has  been  shown  by  a  number  of  investigators,  e.  g.,  Jodidi 
(1909),  Kelley  and  Thompson  (1914),  and  Gortner  (1916  a)  'that 
considerable  amounts  of  nitrogen  are  dissolved  from  certain  soils 
by  this  preliminary  treatment.  Xhe  acid  solution  thus  obtained 
should  contain  the  ammonia,  acid  amides,  amines,  amino  acids,  and 
all  other  organic  nitrogenous  compounds  soluble  in  water  or  very 
dilute  acid.  The  1  per  cent  hydrochloric  acid  extracted  8.57  per  cent 
of  the  total  nitrogen  from  sphagnum-covered  peat,  and  5.09  per 
cent  from  the  calcareous  black  grass-peat. 

Duplicate  nitrogen  determinations  were  made  on  250  cc.  por- 
tions of  the  acid  extract  and  from  these  results  the  total  nitrogen 
in  the  bulk  solution  determined.  The  5500  cc.  solution  from  sphag- 
num-covered peat,  containing  0.6468  gram  nitrogen,  and  the  5000 
cc.  from  the  calcareous  black  grass-peat,  containing  0.4690  grain 
nitrogen,  were  used  for  analysis.  These  solutions  were  concen- 
trated under  reduced  pressure  to  about  200  cc.  and  then  hydrolyzed 
for  48  hours,  after  first  adding  75  cc.  concentrated  hydrochloric 
acid  to  the  solution  from  sphagnum-covered  peat,  and  100  cc.  to 
the  solution  from  the  calcareous  black  grass-peat.  During  evap- 
oration under  reduced  pressure  considerable  hydrolysis  took  place 
for  the  solutions  turned  dark  brown  in  color.  During  hydrolysis 
of  calcareous  black  grass-peat  silicic  acid  separated  in  the  con- 
denser.* 

The  analysis  of  sphagnum-covered  peat  shows  that  over  65  per 
cent  of  the  nitrogen  is  in  the  form  of  ammonia.  Potter  and  Snyder 
(1915  a)  have  shown  that  a  very  small  amount  of  the  nitrogen 
in  the  1  per  cent  hydrochloric  acid  extract  of  soils  exists  in  the  soil 
as  ammonia  nitrogen.  It  seemed  probable  that  if  an  extract  of  the 
peat  contained  so  much  ammonia  nitrogen  after  hydrolysis,  the  air 
dry  peat  must  contain  an  appreciable  amount  in  the  ordinary  con- 
dition. The  ammonia  nitrogen  was  determined  directly  on  a  5  gram 
sample  of  the  air  dry  material.  An  excess  of  calcium  hydroxide 
solution  was  added  and  the  mixture  distilled  under  reduced  pressure 
for  forty-five  minutes.  It  was  found  that  5.40  per  cent  of  the  total 


*This  was  also  true  with  all  of  the  mineral  soils  studied,  and  is  probably 
due  to  the  presence  of  inorganic  fluorides. 


52 

nitrogen  of  the  soil  existed  in  the  form  of  ammonia  nitrogen.* 

The  precipitates  containing  the  "humin"  nitrogen  were 
washed  by  decantation  until  practically  all  the  dissolved  nitrogen 
was  removed.  After  digestion  the  material  was  diluted  to  500  cc. 
and  250  cc.  portions  used  for  distillation.  Before  concentration  the 
filtrate  from  the  "humin"  precipitate  of  sphagnum-covered  peat 
was  reddish  in  color  and  after  concentration  this  color  changed  to  a 
cherry  red.  Twenty-five  grams  of  phosphotungstic  acid  was  used 
for  precipitation  of  the  bases  in  sphagnum-covered  peat.  The 
barmm  phosphotungstate  precipitate  retained  0.0022  gram  nitrogen. 
The  solution  containing  the  basic  nitrogen  was  diluted  to  50  cc. 
and  the  one  containing  total  nitrogen-in-filtrate-from-bases  to  250 
cc. 

The  experimental  data  showing  the  grams  of  nitrogen  found 
and  per  cent  of  total  nitrogen  are  given  in  Table  XVII. 

Table  XVII. — Nitrogen  distribution  of  a  1  per  cent  h\drochloric 
acid  extract  of  sphagnum-covered  peat  and  (in  part)  of  calcareous 
black  grass-peat. 


Sphagnum-covered 
peat 


Calcareous 
black  grass-peat 


Grams 
nitrogen 

Per    cent 
of    total 
nitrogen 

Grams 
nitrogen 

Per  cent 
of    total 
nitrogen 

Total  N 

06468 

04680 

Ammonia.  N               •  • 

04230 

.  55  40 

03013 

64  38 

Humin   N 

00657 

10  16 

00582 

1244 

Basic  N 

00389 

601 

i 

Basic    N    set    free    as    NH3   by    50% 
KOH 

00181 

280 

Basic  N  not  set  free  as  NH3  by  50% 
KOH 

00208 

321 

Amino  N  of  bases  

0.0266 

4.11 

Non-amino  N  of  bases  

0.0123 

1  90 

N  in  filtrate  from  bases  

0.1335 

20.64 

Amino  N  in  filtrate  from  bases  

0.1107 

17.11 

Non-amino  N  in  filtrate  from  bases. 

0.0228 

3.53 

Total  N  regained  

0.6611 

102.21 

1  Distribution    of   remaining'   nitrogen    not    determined. 

14.  Analysis  of  a  portion  of  sphagnum-covered  peat  soluble 
in  4  per  cent  sodium  hydroxide  and  precipitated  by  hydrochloric 
acid  and  (in  part)  of  a  similar  solution  from  a  calcareous  black 
grass-peat.  The  organic  material  soluble  in  4  per  cent  sodium 
hydroxide  was  next  extracted  from  new  portions  of  the  two  differ- 
ent peats.  Twelve  5  gram  portions  were  leached  with  1  per  cent 
of  hydrochloric  acid  to  the  absence  of  calcium  and  the  excess  of 
acid  removed  by  washing  with  distilled  water,  until  the  filtrate 
indicated  only  a  faint  trace  of  free  acid  when  tested  with  Squibb's 
litmus  paper.  After  leaching  and  washing,  each  5  gram  portion 
was  washed  into  tall  glass-stoppered  cylinders  of  500  cc.  .capacity 


*This  was  further  indicated  by  greenhouse  experiments.  The  peat  was 
treated  with  calcium  carbonate  at  the  rate  of  4000  pounds  per  acre  and  planted 
to  barley.  The  plants  made  a  very  rapid  growth  during  the  early  stages  of 
development  and  finally  lodged.  Next  to  this  was  a  plot  of  calcareous  black 
grass-peat  which  contained  only  0.88  per  cent  of  its  total  nitrogen  in  the 
form  of  ammonia  nitrogen.  When  limed  and  sown  to  barley,  it  did  not  show 
any  abnormal  growth. 


53 

with  4  per  cent  sodium  hydroxide  and  filled  to  the  mark.  These 
were  thoroughly  shaken  and  placed  on  their  sides,  thus  allowing 
the  peat  to  settle  on  the  sides  of  the  cylinder,  thereby  exposing  a 
very  large  surface  to  the  action  of  the  hydroxide.  The  shaking  was 
repeated  at  intervals  for  nine  days.  The  cylinders  were  then  thor- 
oughly shaken,  placed  in  a  vertical  position  and  allowed  to  settle 
for  four  days  before  the  supernatant  liquid  was  syphoned  off  and 
filtered.  The  samples  of  calcareous  black  grass-peat  were  almost 
completely  dissolved  by  the  hydroxide  solution. 

These  filtered  solutions  were  neutralized  with  hydrochloric 
acid  (solution  tested  faintly  acid)  when  a  brown  flocculent  precipi- 
tate separated.  This  was  allowed  to  settle  for  several  hours  and 
the  cider  colored  solution  syphoned  off.  The  brownish  black  pre- 
cipitates were  filtered  and  after  draining  over  night  were  thoroughly 
mixed  with  a  large  volume  of  water  and  again  filtered  and  drained. 
The  resulting  precipitates  were  hydrolyzed  with  200  cc.  of  hydro- 
chloric acid  for  48  hours.  This  amount  of  concentrated  acid  was 
added  and  the  flask  brought  to  boiling  until  hydrogen  chloride 
fumes  were  evolved  showing  the  presence  of  constant  boiling  acid. 
Silicic  acid  separated  on  the  condenser  during  the  hydrolysis  of 
calcareous  black  grass-peat.  Even  after  the  hydrolysis  there  still 
remained  some  small  lumps  of  the  "humus"  precipitate. 

The  entire  hydrolysate  was  used  for  the  ammonia  determina- 
tion. After  this  determination  the  "humin"  precipitate  was  thor- 
oughly ground  in  a  mortar  to  insure  complete  disintegration,  al- 
though this  seemed  hardly  necessary  as  the  solid  was  already  in 
a  fairly  fine  state  of  division.  This  precipitate  was  washed  in  the 
usual  manner  by  decantation,  the  filtrate  concentrated  by  evapora- 
tion and  made  to  250  cc.  volume.  Duplicate  portions  of  25  cc. 
were  used  for  the  determination  of  total  nitrogen  in  the  solution 
and  the  result  listed  as  total  nitrogen-in-the-filtrate-from-humin. 
The  remaining  200  cc.  portion  was  used  for  precipitation  of  the 
bases  and  subsequent  analysis. 

The  high  content  of  carbonaceous  organic  matter  made  the 
"humin"  precipitate  very  difficult  to  digest.  The  sulfuric  acid 
required  was  120  cc.  and  the  digestion  extended  over  10  days  be- 
fore complete  decoloration  was  effected.  Of  course  precautions 
were  taken  to  prevent  the  absorption  of  ammonia  from  outside 
sources,  The  material  was  diluted  to  500  cc.  and  250  cc.  portions 
used  in  the  distillation.  The  basic  nitrogen  was  precipitated  with 
25  grams  of  phosphotungstic  acid.  The  nitrogen  retained  by  the 
barium  phosphotungstate  was  0.0043  gram.  The  solution  contain- 
ing the  basic  nitrogen  was  made  to  50  cc.  volume,  and  that  con- 
taining the  total  nitrogcn-in-the-filtrate-from-the-bases  was  made 
to  a  volume  of  250  cc. 

The  experimental  data  showing  the  grams  of  nitrogen  and  per 
cent  of  total  nitrogen  are  given  in  Table  XVIII. 


54 


Table  XVIII. — Nitrogen  distribution  in  that  portion  of  a  sphag- 
num-covered peat  soluble  in  4  per  cent  sodium  hydroxide  and  precipi- 
tated by  hydrochloric  acid  and  (in  part)  of  a  similar  solution  from  a 
calcareous  black  grass-peat. 


Sphagnum-covered 
peat 


Calcareous 
black  grass-peat 


Grams 
nitrogen 

Per    cent 
of    total 
nitrogen 

Grams 
nitrogen 

Per  cent 
of    total 
nitrogen 

Total  N  

0.2860 
0.0464 
0.0950 
0.0309 

0.0070 

0.0239 
0.0174 
0.0135 
0.1175 
0.0965 
0.0210 
0.2898 

0.6344 
0.0781 

0.2093 

i 

Ammonia  N  .  . 

16.22 
33.22 
10.80 

2.45 

8.35 
6.08 
4.72 
41.08 
33.74 
7.34 
101.32 

12.31 
32.99 

Humin  N  

Basic  N  

Basic    N    set    free    as    NH3   by   50% 
KOH  

Basic  N  not  set  free  as  NH3  by  50% 
KOH  

Amino  N  of  bases  

Non-amino  N  of  bases  

N  in  filtrate  from  bases  

Amino  N  in  filtrate  from  bases  

Non-amino  N  in  filtrate  from  bases. 
Total  N  regained  

1  Distribution    of    the    remaining-    nitrogen    not    determined. 

15.  Ana'ysis  of  a  portion  of  sphagnum-covered  peat  soluble 
in  4  per  cent  sodium  hydroxide  and  not  precipitated  by  hydro- 
chloric acid  and  (in  part)  of  a  similar  solution  from  a  calcareous 
black  grass-peat.  The  filtrate  remaining  from  the  brownish  black 
precipitate  formed  by  acidifying  the  sodium  hydroxide  extracts 
of  the  soil  with  hydrochloric  acid  (cf.  section  13)  were  concentrated 
in  the  usual  manner  to  about  700  cc.  when  a  heavy  precipitate  of 
sodium  chloride  separated.  On  standing  over  night  there  also 
separated  a  heavy  flocculent  brown  precipitate.  This  may  have 
been  due  to  the  salting  out  effect  of  the  sodium  chloride  on  some 
of  the  organic  substances  in  the  solution.  The  solution  was  sat- 
urated with  hydrogen  chloride  in  the  cold  and  the  mixture  then 
divided  into  two  portions  and  hydrolyzed  for  48  hours.  The  cold 
material  after  hydrolysis  was  united  and  filtered  through  glass 
wool  and  the  precipitate  washed  with  concentrated  hydrochloric 
acid.  The  filtrate  was  allowed  to  stand  in  a  tall  soil  beaker  when 
more  salt  separated,  due  to  the  increased  concentration  of  the  hydro- 
chloric acid.  The  salt  that  separated  was  freed  from  the  mother 
liquid  by  packing  in  a  centrifuge  and  washing  with  acid  a  number 
of  times.  The  salt  washed  as  free  of  the  solution  as  possible  was 
dried  on  the  steam  bath.  It  was  nearly  white  in  color.  The  glass 
wool  was  dried  and  ground  with  the  salt.  After  being  sampled,  15 
gram  portions  were  used  for  Kjeldahl  determinations.  The  results 
were  listed  as  nitrogen-retained-by-the-salt. 

The  combined  nitrates  were  concentrated  and  analyzed.  After 
the  digestion  of  the  humin  nitrogen,  the  material  in  the  Kjeldahl 
flask  was  made  to  500  cc.  volume  and  the  distillations  carried  out 
on  250  cc.  portions.  The  nitrate  and  washings  from  the  humin 
were  acidified  and  evaporated  under  diminished  pressure  to  less 
than  200  cc.  and  then  made  to  250  cc.  volume.  Duplicate  nitrogen 
determinations  were  made  on  25  cc.  portions  of  the  solution  and 


55 

the  results  listed  as  total  nitrogen-in-the-nltrate-from-humin. 

The  total  nitrogen  in  the  hydrolysate  was  determined  by  add- 
ing the  nitrogen  obtained  as  ammonia,  and  humin  (in  salt  and  that 
precipitated  by  calcium  hydroxide)  to  the  total  nitrogen-in-the- 
filtrate-from-humin. 

Fifteen  grams  of  phosphotungstic  acid  were  used  for  the  pre- 
cipitation of  the  bases.  The  barium  phosphotungstate  from  the 
sphagnum-covered  peat  retained  0.0055  gram  nitrogen.  The  solu- 
tion containing-  the  basic  nitrogen  was  made  to.  50  cc.  vUim"  '•»*  ' 
that  containing  the  total  nitrogen-in-the-filtrate-from-the-bases  was 
made  to  250  cc.  volume. 

The  experimental  data  showing  the  grams  of  nitrogen  and  per 
cent  of  total  nitrogen  are  given  in  Table  XIX. 

Table  XIX. — Nitrogen  distribution  in  that  portion  of  a  sphagnum- 
cohered  peat  soluble  in  4  per  cent  sodium  hydroxide  and  not  precipi- 
tated by  hydrochloric  acid  and  (in  part)  of  a  similar  solution  from  a 
calcareous  black  grass-peat. 


Sphagnum-covered 
peat 


Cateareous 
black  grass-peat 


Grams 
nitrogen 

Per    cent 
of    total 
nitrogen 

Grams 
nitrogen 

Per  cent 
of    totat 
nitrogen 

Total  N 

03736 

06204 

Ammonia  N  ;  .  .  . 

0.0993 

26.58 

0.2003 

32.29 

Humin  N  pptd.  by  Ca(OH)2  
Humin  W  retained  in  NaCl 

0.0335 
00102 

8.97 
273 

0.0652 
0  0169 

10.51 
2.72 

Basic  N 

00324 

867 

2 

Basic    N    set    free    as    NH3   by    50% 
KOH 

00070 

1.87 

Basic  N  not  set  free  as  NH3  by  50% 
KOH 

00254 

680 

\mino  N  of  bases       .  .        

0.0160 

4.28 

Non-amino  N  of  bases 

00164 

4.39 

"V  in  filtrate  from  bases 

0  20061 

5369 

•\mino  N  in  filtrate  from  bases 

0  1721 

4607 

\on-amino  N  in  filtrate  from  bases.. 

0.0285 

7.62 

Total  N  regained  

0.3760 

100.64 

1  Calculated    from   one  determination.      Duplicate   lost    in    digestion. 
8  Distribution  of  the  remaining  nitrogen  not  determined. 

16.  "Jodidi  numbers."  These  determinations  were  carried 
out  as  directed  previously  on  Fargo  clay  loam,  Fargo  silt  loam, 
Hempstead  silt  loam,  prairie-covered  loess,  and  forest-covered 
loess. 

The  resulting  data  in  grams  and  in  per  cent  of  total  nitrogen 
are  shown  in  Tables  XX,  XXI,  XXII,  XXIII,  and  XXIV.  The 
figures  for  similar  fracions  from  the  complete  Van  Slyke  analyses 
are  added  for  comparison.  These  results  will  be  discussed  later. 


56 

Table  XX. — "fodidi  numbers"  determined  on  100  cc.  of  hydrol- 
ysate  of  Fargo  clay  loam,  together  with  a  comparison  of  similar  frac- 
tions taken  from  the  Van  Slyke  analysis. 


Grams  nitrogen 

Per    cent     of    total 
nitrogen 

Average 
data  of 
Van  Slyke 
analysis 

TablfVlIl 

Total  N  
Ammonia  N  
Residue  from  above  acidi- 
fied with  HC1  and  bases 
pptd.  direct,  "Basic  N". 
N  in  filtrate  from  "bases" 
Total  N  regained   

I 

0.0484 
0.0160 

0.0033 
0.0315 
0.0508 

II 

0.0478 
0.0160 

0.0055 
0.0277 
0.0492 

I 

II 

Av. 

33.06 

6.82 
65.08 
104.96 

33.47 

11.51 
57.95 
102.93 

33.26 

9.16 

61.52 
103.94 

24.00 

9.58 
32.71 

Table  XXI. — "Jodidi  numbers"  determined  on  200  cc.  of  hydrol- 
ysate  of  Fargo  silt  loam,  together  with  a  comparison  of  similar  frac- 
tions taken  from  the  Van  Slyke  analysis. 


Grams 
nitrogen 

Per    cent    of 
total 
nitrogen 

Average      data 
of    Van  Slyke 
analysis. 
Table  IX. 

Total  N 

0  1424 

Ammonia  N  

0.0487 

34.20 

26.56 

Residue     from     above     acidified 
with   HC1   and  bases  pptd.  di- 
rect, "Basic  N"  ;  .  .  . 
N  in  filtrate  from  "bases"  
Total  N  regained   

0.0193 
0.0777 
0.1457 

13.55 

54.56 
102.31 

12.11 
35.14 

Table  XXII. — "Jodidi  numbers"  determined  on  100  cc.  of  hydrol- 
ysate  of  Hempstead  silt  loam,  together  with  a  comparison  of  similar 
fractions  taken  from  the  Van  Slyke  analysis. 


Average 

Grams  nitrogen 

Per  cent  of  total 
nitrogen 

Van   Slyke 
ana  ysis 

s 

Table  XI 

Total  N  

I 
00487 

II      1 

0.0513 

I 

11 

Av. 

Ammonia  N 

00207 

00197 

4251 

3840 

4045 

2944 

Residue  from  above  acidi- 
fied with  HC1  and  bases 
pptd.  direct,  "Basic  N". 
N   in   filtrate   from  "bases" 
Total  N  regained  

0.0068 
0.0243 
0.0518 

0.0067 
0.0255 
0.0519 

13.96 
49.90 
106.36 

13.06 
49.71 
101.17 

13.51 
49.81 

103.77 

11.39 
28.10 

57 


Table  XXIII. — "Jodidi  numbers"  determined  on  200  cc.  of  hydrol- 
ysate  of  prairie-covered  loess,  together  with  a  comparison  of  similar 
fractions  taken  from  the  Van  Slyke  analysis. 


Grams 
nitrogen 

Per  cent  of 
total  nitrogen 

Average      data 
of  Van  Slyke 
analysis. 
Table  XII. 

Total  N  

0.0601 

Ammonia  N  .  .          

00244 

4060 

3053 

Residue     from     above     acidified 
with   HC1  and  bases  pptd.  di- 
rect   "Basic  N"  

0.00361 

5.99 

12.68 

N  in  filtrate  from  "bases"  

0.0321 

53.41 

28.80 

Total  N  regained  

100.002 

1  By    difference.        2  Calculated. 


Table  XXIV. — "Jodidi  numbers"  determined  on  200  cc.  of  hydrol- 
ysate  of  forest-covered  loess,  together  with  a  comparison  of  similar 
fractions  taken  from  the  Van  Slyke  analysis. 


Grams 
nitrogen 

Per    cent 
of    total 
nitrogen 

Average      data 
of  Van  Slyke 
analy  sis. 
Table  XIII. 

Total  N  

0.0340 

Ammonia  N  .  .  .      .        .      ... 

0.0130 

3824 

2869 

Residue     from     above     acidified 
with  HC1   and  bases  pptd.  di- 
rect,   "Basic    N"     

0.0059 

1735 

1398 

N  in  filtrate  from  "bases" 

00175 

51  47 

2721 

Total  N  regained  

0.0364 

107.06 

17.  Summary  Tables.  Certain  of  the  preceding  analyses  have 
been  summarized  in  Tables  XXV,  XXVI.  In  Table  XXV  are 
shown  the  amounts  and  percentages  of  soil  dissolved  by  the  acid 
during  hydrolysis  as  well  as  the  amount  of  nitrogen  and  percentage 
of  the  total  nitrogen  dissolved.  Table  XXVI  summarizes  average 
nitrogen  distribution  of  Tables  V,  VI,  VII,  VIII,  IX,  X,  XI, 
XII,  XIII,  XIV,  XVI,  XVII,  XVIII,  and  XIX. 

Table  XXV. — Percentages  of  soil  and  of  soil  nitrogen  dissolved 
by  hydrolysuig  the  different  soil  types. 


Soil  type 

&> 
a 

a 

C/3 

Grams 
soil 
taken 
(dry  basis) 

Grams 
soil 
dis- 
solved 

Per  cent 
soil 
dis- 
solved 

Grams 
nitrogen 
in 
soil 

Grams 
nitrogen 
dis- 
solved' 

Per  cent 
of  dis- 
solved 
nitrogen 

Fargo  clay  loam  

I 

TT 

240.2 
2402 

43.2 
432 

17.99 

0.6005 

0.4252 

70.79 

Kargo  silt  loam 

T 

222.8 

54.8 

24.60 

1.8336 

1.4237 

77.65 

Carrington  silt  loam  .  . 
Hempstead  silt  loam  . 

Prairie-covered  loess  . 
Forest-covered  loess  . 

I 
I 
II 
I 
II 
I 
II 

235.5 
242.3 
242.3 
230.3 
230.3 
294.4 
294.4 

36.5 
32.3 
.  33.3 
42.3 
45.3 
29.4 
32.4 

16.61 
13.33 
13.74 
18.37 
19.64 
9.98 
11.11 

0.8738 
0.6201 

0.6933 
0.3768 

0.6191 
0.4395 
0.4508 
0.5260 
0.4991 
0.2735 
0.2511 

70.91 
70.87 
72.69 
75.91 
72.02 
72.19 
66.65 

aThe  figures  in  this  column  were  obtained  by  subtracting  the  "insoluble 
humin  nitrogen"  remaining  in  the  residual  soil  from  the  nitrogen  figures  ob- 
tained by  multiplying  the  original  weight  of  soil  taken  (dry  basis)  by  the 
nitrogen  content  of  the  soil.  These  figures  may  or  may  not  agree  with  the 
figures  obtained  in  kjeldahling  a  portion  of  the  solution,  due  to  experimental 
errors,  and  perhaps  to  errors  introduced  in  using  a  uniform  factor  (2.6)  for 
specific  gravity.  The  figures  in  this  column  are  free  from  any  error  of  this 
sort. 


paadAOO 


•jl   opiums 


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tUBOl       . 

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•UIBO| 


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PPV 


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.HO«X  %t 


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58 

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59 

18.  An  attempt  to  isolate  pure  proteins  from  a  soil.  From 
all  the  positive  evidence  it  would  seem  that  a  very  considerable 
portion  of  the  nitrogen  in  the  soil  exists  in  other  forms  than  pro- 
tein. For  example.  Potter  and  Snyder  (1916)  found  an  average  of 
20.46  per  cent  of  the  alkali  soluble  nitrogen  of  'a  soil  to  be  non- 
protein  in  nature.  Bacteria  and  fungus  spores  all  contain  chitiri 
which  on  hydrolysis  yields  glucosamine  which  will  give  amino 
(-NH,)  nitrogen  of  non-protein  origin. 

The  average  C/N  ratio  that  exists  in  this  series  of  six  mineral 
soils  studied  has  been  shown  by  Gortner  (1916  a)  to  be  12.23.  The 
average  nitrogen  content  of  the  six  soils  is  0.355  per  cent  which 
would  indicate  2.22  per  cent  protein  (NX6.25)  if  all  of  the  nitrogen 
existed  in  this  form.  However,  the  total  organic  matter  in  these 
soils  averaged  7.48  per  cent  (carbonX  1.724),  showing  that  less 
than  30  per  cent  of  the  soil  organic  matter  could  be  of  protein  na- 
ture. This  is  shown  equally  well  by  a  comparison  of  the  C/N 
ratios.  The  average  analyses  of  sixteen  plant  proteins  as  recorded 
by  Mathews  (1915)  gives  carbon  52.08  per  cent  and  nitrogen  17.73 
per  cent.  The  average  C/N  in  these  vegetable  proteins  is  2.94. 
The  high  C/N  ratio  in  soils  indicates  that  the  organic  matter  does 
not  consist  essentially  of  protein  material  (cf.  however,  Gortner 
1917,  when  a  ratio  of  3.06  was  found  indicating  that  the  organic 
matter  in  this  instance  was  essentially  protein). 

In  view  of  the  desirability  of  demonstrating  the  presence  or 
absence  of  proteins  in  soils  an  attempt  was  made  to  isolate  from 
a  soil  either  alcohol  soluble  or  salt  soluble  proteins.  A  6  inch 
bulk  sample  of  Fargo  silt  loam  from  Morristown,  Rice  County, 
containing  0.397  per  cent  of  nitrogen  in  the  air  dry  soil,  was  used. 

a.  Extraction  with  70  per  cent  ethyl  alcohol.  The  soil  was 
first  leached  with  1  per  cent  hydrochloric  acid  to  the  absence  of 
calcium  and  washed  with  distilled  water  until  practically  all  chlo- 
rfdes  were  removed.  For  this  purpose  sixty-one  100  gram  por- 
tions were  taken.  It  required  about  150  liters  of  acid  to  remove 
all  traces  of  calcium. 

After  leaching,  the  soil  was  allowed  to  air  dry  in  the  green- 
house and  there  remained  5700  grams  air  dry  soil.  Five  hundred 
gram  portions  were  placed  in  twelve  2.5  liter  acid  bottles  and  ex- 
tracted successively  with  70  per  cent  ethyl  alcohol.  A  fresh  por- 
tion of  alcohol  was  added  to  the  first  bottle  of  the  series,  shaken, 
allowed  to  stand  over  night,  syphoned  off,  and  placed  in  the  bottle 
next  ahead  in  the  series.  This  was  continued  until  six  successive 
portions  of  fresh  alcohol  had  been  added.  The  alcohol  was  ab- 
sorbed by  the  dry  soil  to  such  an  extent  that  a  500  gc.  portion  of 
fresh  alcohol  had  to  be  added  to  the  bottles  towards  the  end  of 
the  series.  Almost  15  liters  of  alcohol  were  used,  but  only  a  little 
over  10  liters  were  regained  at  the  end,  since  so  much  was  retained 
by  the  soil. 

The  combined  extracts  were  filtered  until  practically  free  of 
clay  and  then  concentrated  under  diminished  pressure  at  a  tem- 
perature of  55°  C.  The  extracts  were  in  all  cases  straw  colored. 


60 

A  heavy  yellow  precipitate  separated  on  concentration  but  dis- 
solved very  largely  in  the  fresh  portions  of  the  alcohol  extract 
The  greater  portion  of  water  was  finally  removed  by  evaporating 
four  times  with  93  per  cent  alcohol.  More  water,  however,  could 
have  been  removed  by  evaporating  with  absolute  alcohol.  The 
trace  of  clay,  remaining  after  concentration,  was  filtered  off  on  a 
dry  filter  paper  and  the  solution  diluted  to  500  cc.  with  93  per 
cent  alcohol.  The  total  nitrogen  was  determined  on  duplicate  25 
cc.  portions  of  the  solution  by  the  Kjeldahl  method.  The  results 
gave  0.1320  gram  nitrogen  in  the  total  alcohol  extract. 

Tests  were  made  on  portions  of  this  alcohol  extract  for  the 
presence  of  proteins  or  protein-like  substances.  Precipitates  were 
obtained  with  phosphotungstic  acid  and  lead  acetate.  A  heavy 
yellow  precipitate  formed  on  dilution  with  water.  This  yellow 
precipitate  was  readily  soluble  in  sodium  hydroxide  (suggests 
presence  of  acids  or  phenols),  and  in  concentrated  hydrochloric 
acid.  The  biuret  test  and  Liebermann's  reaction  both  gave  nega- 
tive tests,  and  Millon's  reaction  gave  an  extremely  faint  test  (mere 
trace). 

One  25  cc.  portion  of  the  alcohol  solution  was  diluted  with 
water  and  extracted  four  successive  times  with  chloroform,  fol- 
lowed by  a  single  extraction  with  ether.  The  chloroform  and  ether 
extracts  were  combined  and  evaporated  to  dryness  in  a  platinum 
dish  on  the  steam  bath  and  later  in  a  hot  air  oven  at  102°  C.  and 
the  residue  weighed.  The  results  indicated  the  presence  of  0.3108 
gram  of  organic  matter,  making  a  total  of  6.2160  grams  in  the  en- 
tire alcohol  extract. 

After  the  chloroform-ether  extraction  there  remained  a  dark 
brown  granular  insoluble  substance.  This  was  filtered  off  on  a  5 
cm.  Buchner  funnel  and  the  filter  and  precipitate  used  for  the 
determination  of  nitrogen.  The  results  indicated  the  presence  of 
0.0010  gram  maximum  protein  nitrogen  in  this  solution,  leaving 
0.1310  gram  of  non-protein  nitrogen  in  the  same  extract. 

The  negative  color  tests  and  the  small  amount  of  nitrogen 
indicate  that  no  protein  is  present  in  this  soil  that  is  soluble  in 
alcohol. 

Another  25  cc.  portion  of  the  alcohol  extract  was  evaporated 
to  dryness  on  the  water-bath  in  .1  platinum  dish  and  subsequently 
dried'in  an  oven  at  96°  C.  with  a  short  final  heating  at  103°  C.  The 
organic  matter  amounted  to  0.6126  gram,  making  a  total  content 
of  organic  matter  in  the  alcohol  solution  12.2520  grams.  From  the 
data  given  it  was  found  that  only  1.08  per  cent  of  the  organic  mat- 
ter in  the  70  per  cent  alcohol  ext -act  of  the  soil  was  nitrogen. 

b.  Extraction  with  absolute  alcohol.  A  100  gram  portion  of 
the  unleached  soil  was  extracted  with  absolute  alcohol  for  60 
hours  in  a  Soxhlet  extraction  apparatus.  The  extraction  flask  had 
a  capacity  of  500  cc.  and  all  the  joints  of  the  apparatus  were  ground 
glass.  Several  pieces  of  broken  porcelain  were  placed  in  the  flask 
to  prevent  bumping-,  as  the  amount  of  dissolved  organic  matter 
increased.  An  alundum  extraction  thimble  was  used  to  hold  the 


61 

soil.  The  alcohol  syphoning  back  became  colorless  several  hours 
before  the  extraction  was  stopped.  At  the  end  of  the  extraction 
the  solution  in  the  flask  was  deep  straw  color. 

The  total  alcohol  extract  was  evaporated  to  small  volume  and 
then  the  total  nitrogen  determined,  which  was  found  to  be  0.0011 
gram.  This  .was  equivalent  to  0.0660  gram  of  nitrogen  in  6  kilo 
of  soil  before  leaching  with  acid.  It  will  be  recalled  that  0.1320 
gram  nitrogen  dissolved  in  the  70  per  cent  alcohol  after  first  leach- 
ing with  1  per  cent  hydrochloric  acid. 

This  would  indicate  that  many  of  the  organic  nitrogenous 
compounds  are  in  combination  with  the  lime  or  other  bases  present 
in  the  soil,  and  that  this  combination  is  broken  up  when  leached 
with  1  per  cent  hydrochloric  acid. 

c.  Extraction  with  10  per  cent  sodium  chloride.  After  com- 
pletion of  the  alcoholic  extraction,  the  soil  residue  was  dried  in  an 
air  o.ven  at  about  65°  C.  and  placed  in  a  20  liter  bottle.  To  this 
was  added  2000  cc.  of  10  per  cent  sodium  chloride  solution  for 
each  kilo  of  dry  soil.  The  bottle  was  shaken  repeatedly  and  allowed 
to  settle  over  night.  The  presence  of  an  electrolyte  caused  the 
clay  to  settle  rapidly,  so  that  a  clear  solution  could  be  withdrawn. 
After  the  soil  had  been  in  contact  with  the  salt  solution  20  hours, 
two  100  cc.  portions  were  withdrawn  and  analyzed  for  nitrogen. 
The  results  showed  that  0.1350  gram  nitrogen  was  contained  in  .5 
liters  of  the  supernatant  liquid.  After  shaking  and  standing  for 
three  days  duplicate  determinations  were  again  made  on  100  cc 
portions  with  the  result  that  0.1600  gram  nitrogen  was  present  in 
the  corresponding  solution.  This  indicates  that  the  longer  the  soil 
is  in  contact  with  the  sodium  chloride  solution  the  greater  the 
amount  of  organic  nitrogen  extracted. 

Two  500  cc.  portions  of  the  salt  extract  were  used  for  precipi- 
tation with  phosphotungstic  acid.  After  the  addition  of  45  cc.  of 
concentrated  hydrochloric  acid,  the  phosphotungstic  acid  was 
added.  The  gelatinous  precipitates  formed  were  filtered  on  an 
ordinary  funnel  and  washed  with  the  filtrate,  and  then  used  for 
the  determination  of  nitrogen  by  the  usual  method.  The  duplicates 
averaged  0.0028  gram  nitrogen,  thus  making  the  total  nitrogen 
precipitated  by  phosphotungstic  acid  0.0280  gram  in  the  5  liters  of 
supernatant  liquid.  This  shows  that  17.50  per  cent  of  the  nitrogen 
extracted  from  the  residual  soil  was  precipitated  by  phosphotung- 
stic acid.  This  represented  the  maximum  protein  nitrogen  in  the 
10  per  cent  sodium  chloride  solution. 


62 

III.     DISCUSSION. 

A.  Changes  in  nitrogen  distribution  in  a  protein  when  hydro- 
lyzed  in  the  presence  of  a  mineral  soil.  From  a  study  of  Table 
III  it  is  seen  that  the  histidine  nitrogen  formed  when  fibrin  is 
hydrolyzed  alone  is  4.36  per  cent  and  when  hydrolyzed  in  the  pres- 
ence of  cellulose  it  is  4.86  per  cent  of  the  total  nitrogen.  By  re- 
ferring to  Table  II  where  fibrin  is  hydrolyzed  in  the  presence  of 
ignited  subsoil  we  see  the  histidine  nitrogen  is  entirely  lacking  a.nd 
that  the  nitrogen  precipitated  by  calcium  hydroxide  amounts  to 
4.81  per  cent.  This  corresponds  very  closely  to  the  amount  of 
histidine  found  in  the  other  two  cases.  In  other  points  the  three 
analyses  agree  within  experimental  error. 

It  appeared  possible  that  the  histidine  nitrogen  might  have 
been  converted  into  the  nitrogen  fraction  precipitated  by  calcium 
hydroxide.  It  is  a  well  known  fact  that  histidine  can  be  precipi- 
tated by  silver  nitrate  in  slightly  alkaline  solutions.  Since  .there 
are  a  large  number  of  mineral  constituents  in  the  soil  it  may  be 
possible  that  the  histidine  could  be  precipitated  by  some  of  these 
and  thus  be  found  with  the  calcium  hydroxide  precipitate. 

With  this  idea  in  mind  an  analysis  of  histidine  was  made  in 
the  presence  of  an  ignited  subsoil,  only  three  fractions  being  de- 
termined. One  0.5000  gram  sample  of  histidine  di-hydrochloride* 
and  50  grams  ignited  subsoil  were  boiled  in  the  presence  of  100 
cc.  of  hydrochloric  acid  (sp.  gr.  1.18)  for  48  hours.  The  solution 
was  diluted  to  500  cc.  in  a  graduated  flask  and  two  200  cc.  portions 
syphoned  ofif  and  analyzed.  The  solution  was  deep  straw  color. 
The  determinations  for  ammonia  nitrogen  gave  negative  results. 
The  precipitate  formed  by  calcium  hydroxide  was  washed  by  de- 
cantation  until  free  of  dissolved  nitrogen  compounds,  and  the  total 
nitrogen  determined.  This  precipitate  was  bulky  due  to  the  pres- 
ence of  large  amounts  of  ferric  and  aluminum  hydroxides.  The 
average  humin  nitrogen  in  the  two  samples  was  only  0.0006  gram. 

The  filtrate  from  the  calcium  hydroxide  precipitate  was  con- 
centrated to  a  small  volume  and  the  entire  solution  used  for  the 
nitrogen  determination.  Sample  I,  contained  0.0371  gram  in  the 
filtrate,  and  Sample  II,  0.0365  gram,  making  an  average  of  0.0368 
gram. 

The  residual  soil  was  practically  colorless,  and  a  determina- 
tion indicated  that  it  was  nitrogen  free.  The  volume  occupied  by 
the  soil  residue  was  17.3  cc.  By  calculation  it  was  found  that  the 
total  nitrogen  regained  in  the  original  solution  was  0.0903  gram, 
theoretical  0.0921  gram,  or  a  recovery  of  98.01  per  cent. 

Thus  practically  all  of  the  histidine  was  recovered  in  the  fil- 
trate from  the  calcium  hydroxide  precipitate,  indicating  that  the 
hypothesis  was  incorrect. 

*The  histidine  di-hydrochloride  was  prepared  from  dried  blood  as  out- 
lined by  Abderhalden  (1910).  Total  nitrogen  found  18.42  per  cent;  calculated 
18.42  per  cent. 


63 

It  is  possible  that  the  specific  character  of  the  histidine  nitro- 
gen is  destroyed  by  certain  oxidizing  agents  present  during  the 
hydrolysis,  with  the  result  that  histidine  nitrogen  is  not  precipi- 
tated in  the  basic  fraction.  A  study  is  being  conducted  at  the  pres- 
ent time  on  the  hydrolysis. of  pure  protein  in  the  presence  of  certain 
inorganic  oxidizing  agents.  It  is  hoped  that  some  light  will  be 
thrown  upon  the  disappearance  of  histidine  as  well  as  the  formation 
of  "humin"  and  of  the  ''nitrogen  precipated  by  calcium  hydroxide." 

It  is  of  interest  to  note  that  the  average  total  nitrogen  in  the 
two  samples  in  Table  II  is  0.4584  gram  (cf.  Gortner  1916  c,  where 
total  nitrogen  on  four  determinations  of  three  grams  averaged 
0.4551),  showing  that  all  of  the  nitrogen  present  is  accounted  for 
in  the  method  of  analyses  used. 

Table  IV  shows  the  difference  between  the  duplicate  deter- 
minations of  the  analysis  of  fibrin  alone  and  in  the  presence  of  car- 
bohydrate and  of  subsoil,  and  the  differences  apparently  due  to  the 
addition  of  100  grams  ignited  subsoil  to  the  3  grams  of  fibrin.  Van 
Slyke's  (1911)  "maximum"  and  "average"  differences  to  be  ex- 
pected between  duplicate  determinations  are  also  given  in  the  table 
for  reference. 

From  a  study  of  Table  IV  it  will  be  seen  that  the  difference 
between  the  analyses  of  fibrin  hydrolyzed  alone  and  in  the  presence 
of  ignited  subsoil  are,  in  the  case  of  most  of  the  fractions,  within 
the  maximum  allowed  by  Van  Slyke  for  experimental  error.  The 
only  differences  which  are  certainly  greater  than  experimental 
error  are  those  of  humin  nitrogen,  histidine1  nitrogen,  and  amino 
nitrogen  in  the  filtrate  from  the  bases.  It  is  observed  that  prac- 
tically the  same  error  occurred  with  the  amino  nitrogen  in  the  fil- 
trate from  the  bases  when  hydrolysis  was  carried  out  in  the  pres- 
ence of  carbohydrate. 

From  this  analysis  one  can  only  draw  the  conclusion  that  even 
if  the  organic  matter  of  the  soil  consisted  entirely  of  pure  protein, 
one  would  not  obtain  the  same  nitrogen  distribution  by  the  Van 
Slyke  analysis  in  the  presence  of  soil  that  one  would  obtain  in  the 
cibsence  of  the  soil,  or  in  other  words,  the  presence  of  ignitied  min- 
eral subsoil  intefferes  with  the  Van  Slyke  analysis  in  much  the 
same  manner  as  carbohydrates  (Gortner  1916  c,  and  Hart  and  Sure 
1916). 

B.  The  humin  nitrogen,  its  origin  and  significance.  In  such 
a  discussion  one  must  first  consider  the  source  of  humin  nitrogen 
in  pure  proteins. 

Osborne  and  Jones  (1910)  suggest  that  perhaps  tryptophane 
and  histidine  are  responsible  for  the  humin  formation,  basing  their 
pbsttilation  on  the  fact  that  zein,  which  contains  no  tryptophane 
and  but  little  histidine,  gives  only  small  amounts  of  humin  on 
hydrolysis. 

Gortner  and  Blish  (1915)  hydrolyzed  zein  in  the  presence  of 
both  tryptophane  and  of  histidine  and  found  that  a  large  part  of 
the  tryptophane  was  converted  into  humin  nitrogen,  whereas  none 


64 

of  the  histidine  was  converted  into  humin  but  was  all  recoverable 
in  the  bases.  I  have  shown  that  histidine  is  practically  all  recov- 
ered in  the  nitrate  from  the  humin  when  it  is  hydrolyzed  in  the 
presence  of  an  ignited  mineral  subsoil.  Histidine,  therefore,  can 
be  eliminated  as  a  factor  in  the  formation  of  humin  nitrogen  in  the 
soil.  Gortner  and  Blish  conclude  that — 

in  all  probability  the  humin  nitrogen  or  protein  hydrolysis  has  its  origin  in 
the  tryptophane  nucleus. 

Gortner  (1916  c)  has  shown  that  the  humin  nitrogen  is  in- 
creased by  the  addition  of  carbohydrate  material  to  protein,  and 
suggests  that  this  increase  may  be  due  to  both  physical  and  chem- 
ical causes.*  He  presents  evidence  to  show  that  the  action  of  car- 
bohydrate is  probably  due  to  the  furfural  produced  from  the  car- 
bohydrate and  shows  that  increasing  quantities  of  furfural  cause 
the  humin  nitrogen  to  steadily  increase,  and  the  work  of  Gortner 
and  Holm  (1917)  shows  that  the  presence  of  formaldehyde  during 
hydrolysis  causes  a  gradual  increase  in  the  amount  of  humin  nitro- 
gen up  to  a  maximum  very  much  larger  than  the  amount  of  normal 
humin  nitrogen,  and  then  a  decrease  with  increased  amounts  of 
aldehyde. 

Shmook  (1914)  states  that  during  the  hydrolysis  of  his  soils 
there  separated  on  the  walls  of  the  condenser  a  substance  violet  blue 
in  color,  and  that  this  appears  during  the  hydrolysis  of  pure  pro- 
tein an$  is  recognized  as  Liebermann's  reaction  for  protein  sub- 
stances. The  above  conclusion  in  regard  to  the  hydrolysis  of  a 
pure  protein  is  incorrect,  since  no  color  appears  on  the  neck  of  the 
flask  or  condenser  in  such  an  analysis.  When  furfural  is  heated 
alone  with  hydrochloric  acid  a  characteristic  colored  substance  is 
deposited  on  the  condenser.  It  has  been  shown  by  Gortner  (1916 
c)  that  at  the  same  time  a  polymerization  (  ?)  of  furfural  to  humin 
takes  place  very  rapidly.  He  found  that  when,  1.165  grams  of 
furfural  were  heated  with  100  cc.  of  hydrochloric  acid  (sp.  gr. 
1.115)  for  18  hours  that  76.40  per  cent  of  the  original  furfural  was 
converted  into  insoluble  "humin."  Our  mineral  soils  on  hydrolysis 
gave  a  deposit  on  the  condenser  similar  to  that  described  by 
Shmook.  The  reaction  indicates  the  presence  of  furfural,  which 
is  in  turn  formed  from  the  carbohydrates  in  the  soil.  This  must 
be  considered  to  be  a  distinctive  furfural  reaction. 

The  humin  nitrogen  actually  present  in  the  soil  may  easily  be 
a  very  small  part  o-f  the  nitrogen  found.  It  is  evident  that  there 
must  be  many  nitrogenous  organic  compounds  present  in  the  soil 
which  have  no  relation  to  protein  material,  such  as  pu'rine,  pyrimi- 
dine  bases,  nitrogenous  lipins,  and  nitrogenous  pigments  besides 
a  number  of  other  non-protein  substances.  It  is  certain  that  the 
humin  nitrogen  will  be  greatly  changed  by  the  presence  of  many 
of  these  compounds.  The  calcium  hydroxide  here  drags  down  all 
the  organic  nitrogenous  compounds  which  are  soluble  in  dilute 
acids,  but  insoluble  in  hot  water  and  dilute  calcium  hydroxide,  to- 

*Practically  the  same  increase  in  humin  nitrogen  occured  when  fibrin 
was  hydrolyzed  in  the  presence  of  a  mineral  subsoil.  The  humin  in  this 
case  was  not  due  to  the  presence  of  carbohydrate  since  the  soil  had  lost  all  of 
its  organic  matter  by  ignition. 


65 

gether  with  the  calcium  salts  of  nitrogenous  organic  acids,  the 
calcium  salts  of  the  purine  and  pyrimidine  bases  in  addition  to 
the  humin  formed  from  the  protein  material,  and  other  organic 
compounds  that  are  adsorbed,  absorbed,  occluded,  or  combined  with 
the  iron  and  aluminum  hydroxides  present. 

From  Table  XXVI  we  find  that  from  4.84  to  9.21  per  cent  of 
the   total   nitrogen  is  precipitated  by  calcium   hydroxide.     It  can 
readily  be  seen  that  this  does  not  represent  true  humin  nitrogen, 
since  the  calcium  hydroxide  does  not  contain  any  black  colored  sub- 
stances formed  by  hydrolysis.     The  solution  from  which  it  is  pre- 
cipitated is  colored  only  by  ferric  compounds,  therefore,  the  or- 
ganic material  in  this  precipitate  must  consist  of  colorless  organic 
compounds  adsorbed  by  or  combined  with  the  lime.     This  por-  . 
tion  of  the  nitrogen  consists  almost  certainly  of  non-protein  mate- 
rial.    In  all  pure  proteins  the  nitrogen  retained  in  the  calcium  hy- 
droxide precipitate  is  supposed  to  consist  entirely  of  deeply  colored 
compounds.     This  study  of  the.  distribution  of  organic   nitrogen 
in  the  soil  has  led  to  a  new  fraction,  not  previously  reported.     Cer- 
tain of  the  analyses    were    carried    out    before    the    importance 
of  this  fraction  was  realized,  but  in  most  of  the  analyses  I  have 
reported   this    fraction   as   "nitrogen   precipitated   by   calcium    hy- 
droxide," because  of  its  unknown  nature.    Further  investigations  of 
this  fraction  are  highly  desirable. 

Another  point  of  interest  is  observed  in  the  humin  nitrogen  of 
the  sphagnum-covered  peat  hydrolyzed  in  the  presence  of  metallic 
tin.  There  is  a  decided  decrease  in  this  fraction  as  compared  with 
the  peat  hydrolyzed  alone.  As  noted  earlier,  Samuely  (1902),  sug- 
gested that  humin  formation  might  be  due  to  an  oxidation  process 
and  certain  of  the  earlier  workers  (cf.  Hlasiwetz  and  Habermann 
1871  and  1873)  hydrolyzed  protein  in  the  presence  of  stannous  chlo- 
ride in  order  to  obtain  a  colorless  solution  instead  of  one  deeply 
colored  by  the  presence  of  humin. 

I,  therefore,  hydrolyzed  some  gliadin  in  the  presence  of  tin  and 
found  that  while  the  solution  remained  colorless,  nevertheless  small 
balls  of  black  material  were  formed.  The  humin  nitrogen  (insolu- 
ble in  acid  +  that  pptd.  by  Ca(OH)2)  was  1.17  per  cent  of  the  total 
nitrogen,  while  gliadin  hydrolyzed  alone  gave  a  dark  colored  hy- 
drolysate  and  a  humin  nitrogen  content  of  only  0.67  per  cent. 

Recently  Spriestersbach  (private  communication)  has  hydro- 
lyzed fibrin  (from  a  different  sample  than  mine)  alone  and  in  the 
presence  of  stannous  chloride  and  finds  in  the  fibrin  hydrolyzed 
alone  1.67  per  cent  of  total  humin  nitrogen,  of  which  1.06  per  cent 
is  "acid  insoluble"  (cf.  Gortner  1916  c)  and  0.61  per  cent  precipi- 
tated by  calcium  hydroxide.  In  the  sample  of  fibrin  hydrolyzed 
in  the  presence  of  tin  he  finds  0.91  per  cent  of  total  humin  nitrogen, 
of  which  0.25  per  cent  is  "acid  insoluble"  and  0.66  per  cent  pre- 
cipitated by  calcium  hydroxide.  The  nature  of  his  hydrolysate 
agreed  in  all  respects  with  mine,  i.e.,  was  colorless  or  faint  straw 
color,  with  tiny  black  balls  of  humin  floating  on  the  surface  or  at- 


66 

tached  to  the  sides  of  the  flask  in  which  the  hydrolysis  was  carried 
out. 

These  experiments  suggest  interesting  possibilities,  but  dis- 
cussion must  be  deferred  until  we  know  more  of  the  exact  reac- 
tions taking  place. 

The  true  humin  nitrogen  remains  in  the  residual  soil  after 
hydrolysis.  The  amount  of  nitrogen  in  this  fraction  varies  from 
22.93  per  cent  to  28.27  per  cent  of  the  total  nitrogen  for  the  mineral 
soils  studied.  This  represents  more  nearly  the  true  humin  nitrogen, 
in  that  the  black  coloring  matter  formed  by  acid  hydrolysis  remains 
in  this  portion,  but  in  addition  we  should  also  find  here  all  organic 
nitrogenous  compounds  insoluble  in  fairly  strong  solution  of  hy- 
drochloric acid,  all  of  the  nitrogen  adsorbed  by  the  carbohydrate 
humins,  etc.  Potter  an'd  Snyder  (1915  a)  express  surprise  at  the 
large  proportion  of  nitrogen  in  this  fraction  but  when  one  considers 
the  heterogeneous  nature  of  the  soil  organic  matter  it  is  perhaps 
more  surprising  to  find  that  over  60  per  cent  of  the  nitrogenous 
compounds  are  soluble  in  strong  hydrochloric  acid.  Further  study 
is  necessary  before  the  full  significance  and  origin  of  this  humin 
nitrogen  can  be  thoroughly  understood. 

C.  The  effect  of  the  quantity  of  acid  used  for  the  hydrolysis 
on  the  amount  of  nitrogen  dissolved  and  the  nitrogen  distribution 
in  soils.     Throughout  this  investigation  acid  at  least  as  strong  as 
constant   boiling   hydrochloric   acid   was   used   for   the   hydrolysis 
since  that  is  the  strength  recommended  for  the  analysis  of  pure 
proteins. 

In  the  case  of  two  soils,  however,  one  of  the  duplicates  was 
hydrolyzed  in  the  presence  of  1000  cc.  concentrated  acid  to  250 
grams  of  soil,  the  other  being  hydrolyzed  in  the  presence  of  500 
cc.  of  constant  boiling  acid  to  250  grams  of  soil,  in  order  to  see  if 
any  noticeable  differences  would  be  observed  on  the  resulting  analy- 
ses. The  two  soils  thus  hydrolyzed  were  the  prairie-covered  loess 
and  forest-covered  loess. 

The  results  show  little  difference  between  the  duplicates. 
Table  XXV  shows  that  the  larger  volume  of  the  stronger  acid  dis- 
solved a  greater  per  cent  of  the  soil,  due  to  the  fact  that  more  of 
the  mineral  constituents  were  soluble  in  acid  of  this  concentration. 
At  the  same  time,  however,  the  amount  of  nitrogen  extracted  was 
less. 

D.  The  percentage  of  soil  nitrogen  extracted  by  acid  hydro- 
lysis.    Shorey    (1905)    working  with   a   single   Hawaiian   soil   ex- 
tracted 84.68 'per  cent  of  the  total  soil- nitrogen  by  acid  hydrolysis. 
Jodidi    (1911)    working   with    eleven    Iowa    soils   found   from    his 
'studies  a  minimum  of  68.90  per  cent,  a  maximum' of  83.94  per  cent, 
and  an  average  of  75.77  per  cent;  Lathrop  and  Brown   (1911)   in 
five  Pennsylvania  soils  found  a  minimum  of  70.60  per  cent,  a  maxi- 
mum of  73.71  per  cent,  and  an  average  of  71.78  per  cent;  Shmook 
(1914),   working  with   four   Russian   soils,   found   a   minimum   of 
60.60  per  cent  in  the  Laterite  soil,  a  maximum  of  87.67  per  cent  in 
the  Podzol  soil,  and  an  average  of  68.33  per  cent;  Kelley  (1914), 


67 

working-  with  nine  soils  of  the  Laterite  class  common  to  the  Ha- 
waiian Islands,  found  a  minimum  of  67.51  per  cent,  a  maximum 
of  91.80  per  cent,  and  an  average  of  82.17  per  cent;  and  Potter  and 
Snyder  (1915  a)  in  seven  Iowa  soils,  found  a  minimum  of  68.68 
per  cent,  a  maximum  of  76.47  per  cent,  and  an  average  of  74.41 
per  cent. 

The  grand  average  of  all  of  these  thirty-seven  soils  from  wide- 
ly different  origin  gives  75.91  per  cent  of  the  soil  nitrogen  in  solu- 
tion in  the  hydrochloric  acid  extract.  In  my  studies  I  found  a 
minimum  of  66.63  per  cent,  a  maximum  of  77.65  per  cent,  and  an 
average  of  72.19  per  cent  extracted  by  the  acid. 

These  results  indicate  that  the  nitrogen  of  practically  all  soils, 
in  so  far  as  investigated,  dissolves  to  about  the  same  extent  during 
the  acid  hydrolysis. 

E.  "Jodidi  numbers."     A  study  of  Tables  XX,  XXI,  XXII, 
XXIII,  and  XXIV  shows  that  the  nitrogen  distribution  by  this 
method  does  not  give  accurate  results  when  compared  with  similar 
fractions  of  the  Van  Slyke  analyses.     The  ammonia  nitrogen  and 
nitrogen  in  the  filtrate  from  "bases"  are  all  much  too  high,  while 
"basic   N"   corresponds   fairly   well   with   the   true  basic   nitrogen. 
If  one  desires  accurate  data  in  regard  to  the  distribution  of  the 
organic  nitrogen  in  the  soil  he  should  not  use  "Jodidi  numbers." 

F.  Attempts  to  extract  proteins  from  the  soil.     The  attempt 
to  isolate  alcohol  or  salt  soluble  proteins  from  the  soil  was  not  suc- 
cessful.   The  maximum  protein  nitrogen  in  the  70  per  cent  alcohol 
extract  from  6  kilo  of  soil  amounted  to.  only  0.0010  gram,  while 
the  maximum  protein  nitrogen  extracted  by  the  10  per  cent  sodium 
chloride  amounted  to  0.0280  gram  or   17.50  per  cent  of  the  total 
nitrogen  in   solution.     A  larger  amount  of  organic  nitrogen  was 
extracted  by  alcohol  when  the  soil  was  first  leached  with  1  per  cent 
hydrochloric  acid. 

The  amounts  of  possible  protein  were  so  small  that  it  seems 
safe  to  conclude  that  no  appreciable  quantities  of  alcohol  soluble 
or  salt  soluble  proteins  are  found  in  the  soil. 

G.  A  consideration  of  the  nitrogen  distribution  in  different 
extracts  from  the  sphagnum-covered  peat.     Nitrogen  distribution 
was  determined  on  extracts  of  a  sphagnum-covered  peat  soluble 
in  (a)  1  per  cent  hydrochloric  acid,  (b)  4  per  cent  sodium  hydroxide 
and  not  precipitated  by  acidification,  and   (c)   4  per  cent  sodium 
hydroxide  and  precipitated  by  acidification  with  hydrochloric  acid. 
Of  the  three  extracts  only  the  second  approximates  the  distribution 
of  nitrogen  in  a  pure  protein.     The  figures  for  the  ammonia  nitro- 
gen are  abnormally  high  in  the  hydrochloric  acid  extract. 

The  humin  nitrogen  is  high  in  all  the  extracts,  but  is  excessive 
in  fraction  (c).  It  is  clear  that  carbohydrates  from  the  soil  must 
be  present  in  all  three  fractions  used,  and  must  have  some  share 
in  bringing  the  humin  nitrogen  up  to  such  high  figures.  The 
nucleic  acids  (Shorey  191  la,  1912)  would  be  found  in  the  hydro- 
chloric acid  precipitate  from  the  sodium  hydroxide  solution,  and 


68 

the  purine  and  pyrimidine  compounds  of  these  nucleic  acids,  as 
well  as  the  lecithins  (Aso  1904,  Stoklasa  1911)  and  nitrogenous 
lipins  and  nitrogenous  acids  would  be  precipitated  with  the  true 
humin  by  the  calcium  hydroxide. 

The  basic  nitrogen  figures  are  not  widely  divergent  although 
there  may  be  some  significant  differences.  The  differences  be- 
tween the  nitrogen  in  the  filtrate  from  the  bases  is  perhaps  the  most 
significant  of  all.  An  amino  nitrogen  of  only  17.11  per  cent  in  the 
filtrate  from  the  bases  such  as  is  found  in  the  hydrochloric  acid 
extract  is  far  lower  than  has  ever  been  obtained  in  an  analysis 
of  a  pure  protein  and  indicates  that  the  nitrogen  of  this  extract  is 
essentially  non-protein. 

Unfortunately  it  was  impossible  to  complete  the  corresponding 
analyses  on  the  calcareous  black  grass-peat,  but  the  fractions  ob- 
tained would  indicate  a  distribution  similar  to  that  of  the  sphagnum- 
covered  peat. 

H.  General  conclusions  in  regard  to  the  distribution  of  soil 
nitrogen  in  different  soil  types.  From  a  study  of  Table  XXVI  we 
observe  a  great  similarity  between  the  different  fractions.  This  is 
practically  the  same  deduction  made  by  Potter  and  Snyder  (1915  a) 
in  regard  to  their  study  made  on  a  single  soil  type  under  different 
fertilizer  treatment. 

•  I  find  that  the  nitrogen  distribution  in  a  soil  is  very  uniform 
whether  in  the  same  soil  type  under  different  fertilizer  treatment, 
or  in  different  soil  types.  This  is  to  be  expected,  for  if  one  were 
to  take  fifty  Van  Slyke  analyses  of  protein  at  random  and  compare 
the  average  analyses  with  that  of  another  fifty  analyses,  one  should 
expect  to  find  results  agreeing  closely  with  each  other.  This 
expectation  should  also  hold  true  for  the  hydrolysate  of  soils,  since 
in  each  soil  are  to  be  found  many  of  the  nitrogenous  compounds 
contained  in  the  plant  and  animal  products  that  find  their  way 
to  the  soil  together  with  their  decomposition  products.  Since 
there  is  such  a  great  variety  of  different  nitrogenous  substances 
in  the  soil,  it  stands  to  reason  that  the  nitrogen  distribution  in 
soils  is  an  average  distribution,  and  as  such  should  not  be  ex- 
pected to  vary  widely  from  soil  to  soil. 

It  has  been  shown  in  the  earlier  part  of  this  discussion  that 
when  fibrin  was  hydrolyzed  in  the  presence  of  ignited  subsoil,  no 
histidine  fraction  was  obtained. 

For  reasons  which  were  stated  previously,  I  have  not  tabu- 
lated the  nitrogen  distribution  under  the  different  headings  used 
for  the  analysis  of  pure  proteins.  However,  in  view  of  the  results 
obtained  on  the  fibrin  hydrolyzed  in  the  presence  of  ignited  sub- 
soil, it  is  perhaps  worth  while  to  consider  what  values  the  histidine 
fraction  would  have  had.  The  Fargo  clay  loam,  Fargo  silt  loam,  and 
Sample  I  of  the  Carrington  silt  loam,  gave  results  indicating  that 
this  fraction  was  absent,  while  Sample  II  of  Carrington  silt  loam 
gave  2.97  per  cent,  Hempstead  silt  loam  0.78  per  cent,  prairie-cov- 
ered lo.ess  1.21  per  cent,  and  forest-covered  loess  1.25  per  cent  of 
histidine  nitrogen. 


69 

Potter  and  Snyder  (1915  a)  find  this  fraction  to  be  present  in 
all  of  their  eight  soils,  with  a  minimum  of  1.99  per  cent  and  a  maxi- 
mum of  6.30  per  cent.  They  do  not  give  sufficient  analytical  data 
to  permit  a  recalculation  of  their  figures  in  order  to  ascertain  if 
any  errors  in  calculation  were  made.  However,  it  is  possible  that 
their  rinding  is  due  to  the  fact  that  all  of  their  mineral  soils  repre- 
sented a  single  soil  type.  It  is  possible  that  this  form  of  nitrogen 
may  have  been  especially  abundant  in  their  particular  soil.  It  is 
of  particular  interest  to  note  that  their  soil  was  from  the  Wisconsin 
drift  area,  as  was  my  sample  of  Carrington  silt  loam  from  Morris- 
town,  which  gave  my  maximum  amount  of  nitrogen  in  this  frac- 
tion. The  sample  of  Carrington  silt  loam  from  Nerstrand  was  sit- 
uated on  the  Kansan  drift  and  gave  no  "histidine"  nitrogen. 


70 


IV.    SUMMARY. 

This  paper  deals  with  a  study  of  the  nitrogen  distribution  in 
different  soil  types,  by  applying  Van  Slyke's  method.  Tables  have 
been  presented  showing  such  distribution  for  the  following  mate- 
rials : 

a.  Fibrin  hydrolyzed  in  the  presence  of  an  ignited  mineral 

subsoil  (together  with  data  of  fibrin  hydrolyzed  alone 
and  in  the  presence  of  carbohydrates). 

b.  A  calcareous  black  grass-peat. 

c.  An  acid,  sphagnum-covered  peat,  hydrolyzed  alone,  in  the 

presence  of  a  mineral  subsoil,  and  in  the  presence  of 
stannous  chloride. 

d.  An  acid  "muck"  soil 

e.  Seven  samples  of  mineral  surface  soil  representing  the  fol- 

lowing soil  types :  Fargo  clay  loam,  Fargo  silt  loam, 
Carrington  silt  loam  (two  samples  from  different  glacial 
drifts),  Hempstead  silt  loam,  prairie-covered  loess,  and 
forest-covered  loess. 

f  .  Extracts  of  a  sphagnum-covered  peat  and  of  a  calcareous 
black  grass-peat  soluble  in  (a)  1  per  cent  hydrochloric 
acid,  (b)  4  per  cent  sodium  hydroxide  but  precipitated 
by  acid,  and  (c)  4  per  cent  sodium  hydroxide  and  not 
precipitated  by  hydrochloric  acid. 

The  following  conclusions  are  evident : 

1.  The  figures  for  the  ammonia  nitrogen  in  a  protein  analysis 
are  not  appreciably  changed  when  the  hydrolysis  is  carried  out  in 
the  presence  of  an  ignited  mineral  soil  equal  to  twenty  times  the 
weight  of  the  protein  material. 

2.  The  "humin"  nitrogen  is  greatly  increased  by  the  addition 
of  ignited  mineral  soil.    It  was  shown  that  histidine  nitrogen  cannot 
account  for  this  increase,  neither  is  it  due  to  the  presence  of  car- 
bohydrates, since  the  soil  lost  all  its  organic  matter  on  ignition. 

3.  Attention  has  been  called  to  the  fact  that  the  analysis  of 
a  pure  protein  in  the  presence  of  even  an  ignited  mineral  soil  docs 
not  give  reliable  results  for  the  different  fractions,  and  that  such 
determinations  are  of  value  only  when  used  for  purposes  of  com- 
parison.    Such  data  should  not  be  compared  with  analyses  ot  pure 
proteins. 

4.  Since  practically  all  mineral  soils  give  furfural  on  treat- 
ment with  acid  it  is  very  likely  that  a  very  considerable  amount 
of  the  total  humin  nitrogen  found  is  due  to  the  presence  of  carbohy- 
drates in  the  soil,  which  give  rise  to  furfural  during  hydrolysis. 
This  may  combine  with  certain  of  the  nitrogenous  compounds  and 
cause  an  increase  in  the  "humin"  nitrogen,  as  well  as  adsorb  or 
occlude  nitrogenous  compounds  in  the  "humin"  formed  from  fur- 
fural by  polymerization. 


71 

5.  This  investigation  of  the  distribution  of  organic  nitrogen 
in  the  soil  indicates  a  new  fraction,  the  nature  of  which  has  not 
been  previously  recognized.     This  is  the  fraction  of  nitrogen  re- 
moved from  a  colorless  solution  by  calcium,  iron,  and  aluminum 
hydroxides  on  the  addition  of  calcium  hydroxide.     The  nitrogen 
retained  in  this  fraction  must  consist  almost  entirely  of  non-pro- 
tein material,  since  the  organic  substances  in  "this  precipitate  have 
been   shown   to  be   colorless  organic   compounds   adsorbed   by  or 
combined  with  the  metallic   hydroxides.     This   fraction   has  been 
reported  as  nitrogen  precipitated  by  calcium  hydroxide. 

6.  The  true  humin  nitrogen  remains  in  the  residual  soil  after 
hydrolysis,   but   in   addition    non-humin     nitrogenous     compounds 
must  also  be  retained  in  this  fraction. 

7.  The  strength  and  volume  of  the  hydrochloric  acid  used  in 
hydrolysis  has  little  effect  On  the  nitrogen  distribution  of  the  hy- 
drolysate  provided  acid  as  strong  as  constant  boiling  acid  is  used, 
in  the  proportion  of  at  least  two  parts  of  acid  to  one  of  soil. 

8.  Results  gained  from  a  study  of  different  soils -indicate  that 
the  organic   nitrogen   dissolves,   during   hydrolysis,   to   almost   the 
same  extent  regardless  of  the  origin  and  nature  of  the  soil. 

9.  Some  very  interesting  figures  are  found  in  the  comparison 
of   the   different   extracts    from     sphagnum-covered     peat     (Table 
XXVI).     The  portion  soluble  in  sodium  hydroxide  and  not  pre- 
cipitated  by   hydrochloric   acid   gives   a   nitrogen   distribution   ap- 
proximating very  closely  that  of  a  normal  plant  protein.    The  nitro- 
gen dissolving  in  the  preliminary  hydrochloric  acid  leaching  shows 
a  nitrogen  distribution  which  is  certainly  not  due  exclusively  to 
protein  materials,  e.  g.,  an  ammonia  nitrogen  percentage  of  65.40 
and   amino-nitrogeii-in-filtrate-from-bases   of   17.11    per  cent. 

10.  When  an  attempt  was  made  to   isolate  alcohol   soluble 
and  salt  soluble  proteins  from  the  soil  the  amounts  obtained  were 
so  small  that,  it  seems  safe  to  conclude  that  no  appreciable  quan- 
tities of  these  types  of  proteins  are  present. 

11.  The  most  significant  fact  brought  out  by  this  study  is 
that  the  organic  nitrogen  distribution  in  different  soil  types  is  very 
uniform.    This  is  to  be  expected  since  it  has  been  pointed  out  that 
the  nitrogen  distribution  in  soils  is  an  average  distribution  of  all 
the  plant  and  animal  nitrogenous  products  that  find  their  way  to 
the  soil. 


72 


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BIOGRAPHICAL. 

Clarence  Austin  Morrow  was  born  near  Morrow,  Warren 
County,  Ohio.  He  graduated  from  the  Hillsboro,  Ohio,  high  school 
in  June,  1901.  He  entered  the  Ohio  Wesleyan  University  in  the 
Jail  of  1902,  and  received  the  degree  of  Bachelor  of  Science  in  June, 
1906.  During  1906-08  he  held  the  position  of  Assistant  in  Chem- 
istry at  Oberlin  College.  He  received  the  degree  of  Master  of  Arts 
from  the  same  institution  in  June,  1909.  In  1909-10  he  was  acting- 
head  of  the  Departments  of  Chemistry  and  Physics  at  Doane  Col- 
lege. Having  been  appointed  the  John  Harrison  Scholar  in  Chem- 
istry at  the  University  of  Pennsylvania  he  entered  that  institution 
in  the  fall  of  1910  and  continued  his  graduate  work.  In  the  spring 
of  1911  he  was  appointed  John  Harrison  Fellow  in  Chemistry,  but 
later  resigned  this  to  take  the  position  of  Professor  of  Chemistry 
at  Nebraska  Wesleyan  University,  which  position  he  has  held  to 
date  (January,  1917).  During  1914-15  he  was  granted  leave  of 
absence  for  study  in  the  Division  of  Soils,  University  of  Minnesota, 
where  he  held  the  position  of  Assistant  in  Soil  Chemistry.  Here 
he  studied  for  the  degree  of  Doctor  of  Philosophy. 

Major  subject,  soil  chemistry. 

Minor  subject,  organic  chemistry. 

Member  of  the  American  Chemical  Society. 

Member  of  the  American  Association  for  the  Advancement  of 
Science. 


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